Directing Parthenogenetic Stem Cells Differentiate Into Tenocytes For Tissue-engineered Tendon Regeneration

The critical function of tendon is to transfer force from muscle to bone to support body movement. Tendon injury due to acute or chronic trauma are common disorders and may cause pain and body morbidity in both professional and private life. Injured tendon has a limited self-healing capacity due to its avascularity and acellularity. Currently, the clinical options to treat tendon injuries are autografts and allografts transplantation. However, harvesting of autografts will create a secondary morbid site, and allografts transplantation face the risk of infectious pathogens transmission [1-3]. Furthermore, transplanted grafts rarely restore structural and functional integrity of injured tendon due to poor healing at tendon-tendon or tendon-bone interface.Cell therapy based approach, tissue engineering for example, showed great potential for tendon regeneration and repair. Considerable efforts have been made to employ various types of cell for tendon regeneration and repair. But up to now no clinically practicable approach has been developed. Tenocyte is an adequate cell source for tendon regeneration. Despite donor site morbidity, limited number of cells could be obtained from the explanted tissue because tendons are relatively acellular [4], and increasing passage number may result in t phenotypic drift[5]. Dermal fibroblasts are more readily available through a simple dermal biopsy and amenable to culture. Dermal fibroblasts could adhere and proliferate on biomaterials, and synthesize many components of ECM. The key concern about fibroblast is scar formation, which may significantly influence the mechanical properties of tendon. Mesenchymal stem cells (MSCs) are characterized by their extensive proliferative ability and the potential to differentiate along various lineages including bone, cartilage, adipose and tendon. Previous studies showed that MSCs could form tendon like tissue and improved mechanical properties of injured tendon. But long term in vivo observation demonstrated that ossification frequently occurred in the repaired tissue, which may impair the structure and function of tendon. Ouyang’s group established a practical strategy of stepwise differentiating ESCs for in vitro and in vivo tendon regeneration. The results suggest that the progenitor cells derived from ESCs promote tendon regeneration by secreting fetal tendon-specific matrix and differentiation factors. Furthermore, no teratoma and ossification could be observed in the newly form tissue[6]. However, harvesting ESCs involves the destruction of viable embryos, and this strategy still needs to face political and ethical limitation.Parthenogenesis refers to embryonic development of eggs activated artificially without fertilization. Parthenogenetic stem cells (pSC) could be obtained from the inner cell mass of blastocyst. In primates, parthenotes are unable to grow into viable fetuses, because genetic defect affects proper placenta formation. pSC are more histocompatible due to the presence of homozygous HLA genotypes. These common HLA haplotype matched pSC may reduce the risk of immunerejection after transplantation of their differentiated derivatives, thus offering significant advantages for application to cell-based therapies over ESC derived from fertilized oocytes having unique sets of HLA genes. pSCs can develop into retinal pigment epithelium-like cells[7], muscle-like and bony-like cell[8], neuronal cell[9,10] and hepatocyte[l 1]. It was reported that the cardiomyocytes derived from pSCs could be obtained to facilitate engineering of myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial damage. Importantly, it was also showed the immunological acceptance of MHC-haploidentical pSC allografts in related and unrelated recipients [12]. However, no studies to date have identified the capacity of tenogenic differentiation with pSCs.Methods1. Cell culturepSCs (C57BL/6 strain) and ESCs (J1; 129S4/SvJae strain) were cultured on mitotically inactivated mouse embryonic fibroblasts and maintained in Esgro Complete Plus.pSCs and J1 cells were cultured on glass coverslips coated with gelatin and then fixed with 4% paraformaldehyde in PBS for 1 h at 4℃. Primary antibodies used were as follows: anti-Nanog, anti-OCT4, anti-SSEA-1, anti-vimentin, anti-CD34, anti-Sca-1, anti-N-cadherin, anti-E-cadherin, anti-Myog, anti-Nt5e, anti-a-actin.The cells were dissociated with Accutase (Millipore) and resuspended to obtain single-cell suspensions of pSCs and Jl. After that, the cells were counted and seeded into six well plates at four densities to form colonies in Esgro Complete Plus medium in triplicate for each cell density without feeder layer. After 5 days, the formed colonies were stained with 1% crystal violet and counted.Cell viability was analyzed using WST-1 assays. For this procedure, cells were seeded in 96-well plates at different cell densities from 1000 to 2000 cells/100 μL in Esgro Complete Plus medium in quintuplicate for each cell density. A 10-μL volume of cell proliferation reagent WST-1 was added to each well, and plates were incubated at 37℃/5% CO2 in a humidified incubator for up to 60 h.Cellular AP activity was detected using an AP Kit following the manufacturer’s instructions.Cells were visualized using a phase contrast microscope throughout culture. For TEM, prepared samples were sectioned to 70-90-nm thicknesses. Ultrathin sections were stained in 3% aqueous uranyl acetate and then in Sato triple lead stain prior to examination using an FEI CM12 Electron Microscope.2. EB formation and TUNELThe pSCs and J1 cells were dissociated with Accutase (Millipore) and resuspended in cell growth medium. To explore whether pSCs could form EBs and to examine the EB formation efficiencies of pSCs and J1 cells, we used limiting dilution to place one cell into separate wells of ultra-low attachment 96-well plates. Each well was examined to verify the number of cells 2 day after plating. The tightly packed spheres were identified as EBs and counted 5 days later. The 5-day EBs were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, and sectioned. Representative sections were processed for H&E staining and TUNEL assay.3. Spontaneous differentiation in adherent cultureThe 5-day EBs were plated onto dishes coated with 0.1% gelatin to evaluate their developmental potential by monitoring the expression of specific markers. EB outgrowths were allowed to spontaneously differentiate in medium for 28 days with medium changes every 3-4 days.Total RNA was extracted from the cells using an RNA Isolation Reagent according to the manufacturer’s protocol. The extracted RNA was quantified using a GeneQuant pro. A RevertAid First Strand cDNA Synthesis Kit was used to convert the RNA template into cDNA. qPCR was performed using a Bio-Rad real-time PCR system. The relative levels of gene expression were normalized to the β-actin gene using the comparative CT method according to the manufacturer’s instructions.To evaluate whether pSCs possessed pluripotent differentiation capacity in vivo, the cells were dissociated with Accutase (Millipore), and 1 × 106 cells resuspended in 50 μL DMEM were injected subcutaneously into nude mice. After 3-4 weeks, the formed teratomas were harvested and fixed in 4% paraformaldehyde, dehydrated through a graded series of ethanol, embedded in paraffin, sectioned, stained with H&E, and observed.4. Derivation and expansion of pMSCsThe 5-day EBs were plated onto dishes coated with 0.1% gelatin and cultured with CGM for 7 days with medium changes every 3 days. Spindle-shaped cells were observed in the outgrowths. The cells were then selectively separated by cell scrapers and leave the scrapes to sediment at the bottom of the tube, subcultured in MesenCult MSC Basal Medium for 21 days. The culture medium was changed every other day. Cells were subcultured for additional 2-3 passages before use.pMSCs and eMSCs (passage 3) were dissociated and washed with PBS. We immunolabeled 1 × 105 cells with 1 mg of PE-or FITC-conjugated rat anti-mouse monoclonal antibodies against CD13, CD29, CD34, CD44, CD45, CD90.2, CD105, CD117 and CD133 for 1 h at 37℃. For negative controls, isotype-matched negative control antibodies were used under the same conditions. After three washes, the cells were examined using a FACSCalibur cytometer, and the data were analyzed with Cell Quest software.5. Induction toward multiple cell lineages and special stainingpMSCs and eMSCs were induced to differentiate into osteogenic, chondrogenic, and adipogenic lineages. For osteogenic differentiation, both types of cells were cultured in osteogenic medium. The medium was changed every 3 days. After 3 weeks, the cells were fixed in 4% paraformaldehyde and processed for alizarin red S and Von Kossa staining. For chondrogenic differentiation, cells were cultured in chondrogenic medium. Cells were processed for Safranin O and immunocytochemistry staining for Col2al and Aggrecan after 3 weeks. For adipogenic differentiation, cells were exposed to adipogenic medium for 4 weeks. The medium was changed every 3 days. After 4 weeks, the cells were processed for Oil Red O and immunocytochemistry staining of AP2 and C/EBP.6. Mechanical stretch stimulationMechanical stretch was applied to induce cellular tenogenic lineage differentiation with the Flexcell FX-5000 Tension System. pMSCs, dermal fibroblasts, and BMSCs were seeded onto collagen type I-coated BioFlex plates at a density of 1 × 105 cells/well. When the cultures reached approximately 70%-80% confluence, the cells were subjected to cyclic mechanical stretch with 10% elongation for 24 h or 10 days (16 h/day). Each cycle consisted of a 10-s stretch and 10-s relaxation. Control cultures were grown under the same conditions but without stretch.pMSCs were subjected to cyclic mechanical stretch with 10% elongation for 24 h or 10 days (16 h/day). Total protein was quantified using a BCA Assay Kit (Thermo). Proteins were detected using the ECL Advance chemiluminescent substrate. The primary antibodies used were as follows:anti-Col1A1, anti-Col3A1, anti-Tnmd, anti-SCX, anti-GAPDH, and anti-EYA2.7. In vitro evaluation of cell growth on scaffoldsCells were labeled with CM-Dil (Invitrogen) according to the manufacturer’s instructions before seeding into PLGA scaffolds to track cells in vitro and in vivo.For in vitro evaluation of cell growth,1-cm crimp-like poly(lactic-co-glycolic acid) PLGA (Ethicon, LA/GA 10:90)scaffolds were sterilized with 75% ethanol and air dried before use. Next,1 × 105 pMSCsi (pMSCs after 10 days of stretch) in 10 μL medium were then seeded onto scaffolds in six-well plates. Three specimens were harvested at predesignated time intervals of 5,10, and 15 days. DNA was isolated from each construct with DNAiso reagents according to the manufacturer’s protocol. The extracted DNA was quantified using a GeneQuant pro.After observation by laser confocal microscope, three specimens from each scaffold group at different time intervals were prepared for SEM examination. The specimens were prefixed with 2% glutaraldehyde, postfixed with 1% osmic acid, sputter-coated with gold, and examined with a scanning electron microscope to observe cell attachment. 8. In vivo transplantation and observationpMSCs’-PLGA constructs were transplanted subcutaneously into the backs of 6-week-old nude mice. The constructs were harvested at 6 and 12 weeks after implantation for histological (H&E staining and Masson’s trichrome staining), immunohistochemical, and electron microscopic examination. For immunohistochemical analysis, the sections were immunolabeled with primary antibodies, including Col1A1, Col3A1, Col5A1, TNC, Tnmd, SCX, Hsp47, EYA2, BMP2, and BMP13. Isotype-matched negative control antibodies were used under the same conditions as negative controls..Result1. Characterization of pSCsThe commonly used criteria to define stem cells are pluripotency, clonogenicity, and self-renewal. pSCs formed compact, saucer-shaped colonies and expressed high level of alkaline phosphatase (AP) activity. Transmission electron microscopy (TEM) showed that the undifferentiated ESCs had large nuclei, a high nucleocytoplasmic ratio, dense pools of glycogens within the scant cytoplasm, and few mitochondria. pSCs also stably expressed pluripotent markers, including SSEA-1, Nanog, and OCT4. A small population (-14-20%) of pSCs formed adherent cell colony. exhibited dot-and ring-patterned clones. qRT-PCR results indicated that J1 cells exhibited. higher expression levels of vitronectin(VTN) (3.16 ± 0.56 versus 1.09 ± 0.20, respectively; p < 0.001), had no significant difference in β integrin-1 (ITGB1) expression.WST-1 assays showed that Jl cells proliferated more rapidly than pSCs at all densities, as assessed at six time points. To analyze the molecular basis of these patterns of proliferation, we examined the expression of the imprinted cell-cycle regulatory factors insulin-like growth factor 2 (IGF2; maternally imprinted), IGF2r, and p57KIP2 (paternally imprinted) using a continuous passage procedure (with 12 passages). The results demonstrated that pSCs maintained low expression of the igf2 gene (1.09 ± 0.21 versus 3.26 ± 0.4, respectively; p<0.001), may result in a decline in proliferative rates.2.Embryoid body (EB) formation and apoptosisAt 5-7 days after plating in nonadherent dishes, tightly packed spheres (EBs) had formed and were counted. The results showed that J1 cells formed EBs with an efficiency of 35.3% ± 5.2%, while pSCs formed EBs with a lower efficiency of 21.7% ±3.4%.Parts of EBs were fixed on day 5 and were then embedded in paraffin for hematoxylin and eosin (H&E) staining and TUNEL assays. On day 5, the percentage of apoptotic nuclei in EBs derived from pSCs was similar to that derived from J1 cells (9.28% ± 3.8% versus 8.79% ± 2.21%, respectively; p= 0.728). This confirmed that the machinery necessary for apoptosis is inherited from the oocyte. And apoptosis plays a key role in the generation of many cavitated structures in embryos occur and EBs.3.pSCs possessed pluripotent differentiation capacity in vitro and in vivoEBs were seeded on gelatin-coated culture plates, and the morphology of EBs and their outgrowth were observed every other day. At 2 days after plating, the spheres became flattened, and outgrowth of brick-shaped cells, beating cardiomyocytes, and spindle-shaped cells was observed during culturing. We found that the molecular profiles of pSCs and J1-derived cells exhibited similar characteristics toward ectodermal, mesodermal, and endodermal lineage differentiation by qRT-PCR. Immunocytochemistry studies further confirmed the expression of early mesodermal (Sca-1 and CD34) and mesodermal (Nt5e, Myog, vimentin, a-actin) markers at 7 or 14 days after EB plating. qRT-PCR and immunocytochemistry studies showed that the process was accompanied by gained expression of snail1, snail2, vimentin, n-cadherin and lost e-cadherin, suggesting that the differentiation process of pSCs was also associated with EMT (epithelial-mesenchymal transition) conversion. Histological observation showed that pSCs were able to form teratomas containing three germ layers of mucous glands (endoderm); cartilage, osteoblast, muscle cells, and lymph vessels (mesoderm); and neurocytes (ectoderm).4. Generation of MSCs from pSCs and J1 cellsAccording to gene expression levels for mesodermal commitment, the outgrowth of spindle-shaped cells was selectively isolated with a cell scraper 7 days after EB plating, and the scraped cells were subsequently expanded in MesenCult MSC Basal Medium to obtain MSCs. Ultrastructural investigations showed that the mitochondria had dense matrices and tubular cristae, with decreased nucleocytoplasmic ratios. Structural filaments could be observed around the nucleus.pMSCs and embryonic MSCs (eMSCs) had similar surface marker expression profiles. The pMSCs expressed CD29 (b1 integrin), with the majority of cells expressing CD44 (hyaluronate receptor; 56.14%), CD90.2 (thymocyte differentiation antigen-lb; 55.22%), and CD105 (endoglin, SH2; 72.47%). CD13 (alanyl aminopeptidase) was expressed in 23.02%of cells. Immunocytochemistry staining showed that the pMSCs consistently expressed Myog, MHC, Vimentin, and α-Actin.5. pMSCs were capable of osteogenic, chondrogenic, and adipogenic differentiationThe multidifferentiation potential of pMSCs toward osteogenic, chondrogenic, and adipogenic lineages was analyzed. qRT-PCR analysis showed that the expression of osteogenic markers, including alkaline phosphatase (alp), bone sialoprotein (ibsp), osteocalcin (bglapl), secreted phosphoprotein 1 (opn), and runt-related transcription factor 2 (runx2), were increased. Strong staining for Alizarin Red S and Von Kossa 21 days after induction demonstrated accumulated mineralization, indicating that these cells had osteogenic potential after induction.Safranin O, immunocytochemistry staining, and qRT-PCR verified the chondrogenic lineage differentiation. The cells also had the capacity to undergo adipogenic differentiation. This was evident through the accumulation of lipid vacuoles and upregulated expression of the adipogenic markers ap2 and c-ebp after induction.6. Mechanical stimulation enhanced the tenogenic phenotype of pMSCsTo determine whether pMSCs were tenogenic, we used a mechanical stimulation assay. Dermal fibroblasts, bone marrow stem cells (BMSCs), and patellar tenocytes (PTCs) acted as controls. We found that the ratio of colla1:3a1, which is considered as an important indicator for tenogenic differentiation, decreased in BMSCs and PTCs during progressive passaging, especially in PTCs. In contrast, the ratio remained stable for fibroblasts and pMSCs. After a short-term 8-h stretching, the expression levels of collal, col3a1, tnmd, and eya2 genes exhibited an approximate 8-fold upregulation, whereas no substantial upregulation of scx was observed. Western blot analysis also showed that expression of the tenogenic markers Collal, Col3a1, Tnmd, and Eya2 was increased after mechanical stimulation for 24 h.After exposure to 10% mechanical stretching for 10 days, the same trend was observed in samples stretched for 24 h. The expression of collal, col3a1, tnmd, eya2 and scx genes was upregulated by about 20-fold; the increased expression of proteins Collal, Col3a1, Tnmd, and Eya2 was confirmed by western blot analysis.Immunocytochemistry results confirmed that mechanical stretching significantly enhanced collagen biosynthesis (as shown by increased expression of Collal, Col3a1, Col5a1, and Hsp47), induced tenocyte formation, and promoted tenocyte healing (as shown by increased expression of BMP2 and BMP 13) and the tenocyte phenotype (as shown by increased expression of SCX, Tnmd, EYA2, and TNC). Additionally, mechanical stretch had no significant influence on cell proliferation at the end-point of loading.7. pMSCs1 could proliferate on PLGA scaffoldsAt 10 days after mechanical loading, pMSCs (termed here pMSCs1) were seeded on PLGA fibers and cultured for 15 days in vitro. CM-Dil staining and scanning electron microscopy (SEM) observations indicated that pMSCsi adhered to and proliferated well on PLGA fibers. We found that cells bridged the fibers, covered the surface of the scaffolds and began to secrete fine collagen fibrils 15 days after seeding. DNA content increased steadily during in vitro incubation. These results indicated that the PLGA scaffold was suitable for pMSCsi cultivation.8. pMSCs1 regenerated tendon tissue in vivoWe examined the ability of pMSCsi to regenerate tendon tissue in vivo. All animals survived throughout the experiment. The animals were sacrificed at weeks 6 and 12 after implantation in order to harvest specimens. Gross inspection showed that the newly formed tendon had a smooth and glistening white surface. Histology results showed that there was no teratoma formation after implantation. At 6 weeks, H&E staining revealed the tissue containing a large number of spindle-shaped cells, which were situated along the axis of PLGA fibers. Masson’s trichrome staining revealed some positively stained area, indicating collagen deposition in the construct. PLGA scaffolds were partially degraded. TEM observations showed that some fine collagen fibers, having an average diameter of 42.48 ± 9.69 nm, formed in the construct.In contrast, the number of cells in newly formed tendon tissues was significantly lower at 12 weeks than at 6 weeks postoperation. H&E and Masson’s trichrome staining revealed a structure of longitudinally aligned fibers and cells with increased matrix deposition. Typical crimp-patterned collagen fibers with proper cell density could be observed. TEM observations further showed the formation of wave-like collagen fibers, having an average diameter of 54.77 ± 8.95 nm, in the specimen. Most of the PLGA scaffolds were degraded.The red fluorescent signal of CM-Dil-labeled cells was distributed in the newly formed tissue. Immunohistochemistry staining confirmed that the engineered tendon sustained expression of tendon-specific markers, including CollAl, Col3A1, Col5A1, TNC, Tnmd, SCX, Hsp47, EYA2, BMP2, and BMP13. ConclusionWe demonstrate that pSCs exhibit properties similar to ESCs, including pluripotency, clonogenicity, self-renewal and the ability of in vitro and in vivo differentiation. pSCs could be induced to differentiate into parthenogenetic mesenchymal stem cells(pMSCs) and further developed into tenocytes through cyclic mechanical stimulation. Finally we engineered tendon-like tissues in vivo with the induced tenocytes. The newly formed tissues possessed the structural properties of native tendon and expressed tendon-specific marker. Collectively, the study indicates that pSCs are an attractive cell source for tendon tissue regeneration.