Mimicking Stem Cell Niche For Amplification Of ESCs And Construction Of Skin Substitute In One-step

1. BackgroundEpidermal stem cells (ESCs) are mainly found in the stratum basale and hair folliclebulge. But as ESCs are small in number and proliferate slowly, how to amplify themrapidly has been a crux and focus of attention in skin tissue engineering research. Atpresent, ex vivo culture and amplification of ESCs are mainly achieved by spreadingtype-IV collagen (Col IV), the main component of the basement membrane, on the Petridish in a2D mode by taking advantage of the adhesive property of ESCs to the basementmembrane. However, it is difficult to obtain a sufficient amount of amplified ESCs withina short period. In addition, ESCs would differentiate gradually and lose the properties ofstem cells after multiple passages and amplifications.Recently, some researchers used microcarrier culture technique to amplify stem cellsand got good results. For example, they used Cultispher-S, Cytodex, Solohill andMatrigel-coated cellulose to amplify mesenchymal and embryonic stem cells, thus assuringto obtain large amounts of amplified stem cells within a short period without losing theirpotential of multi-directional differentiation. The microcarrier culture technique wouldpromisingly provide a3D space more suitable for the growth and proliferation of ESCs.However, most microcarriers available at present are made of artificially synthesizedbiomaterials. Despite the use of surface modification or decoration techniques, thesemicrocarriers lack the basement membrane structure and thus are unable to mimic the stemcell niche microenvironment for adhesion and proliferation of ESCs.Studies have demonstrated that human amniotic membrane (AM) is an ideal carrierfor cell culture and amplification, because it consists of the thickest human naturalbasement membrane and is structurally similar to the basement membrane of the skin andcornea. In addition, the matrix of human AM contains abundant growth factors such as NGF, HGF, KGF, bFGF, TGF-β1and EGF, which can mimic the stem cell niche for exvivo growth. For this reason, human AM has been used for amplification andtransplantation of limbal and mesenchymal stem cells.In the present study, we created a3D micronized AM (mAM) with the intact structureof basement membrane and bioactivities by taking advantage of the intrinsic biologicalproperties of AM, hoping to use it as a novel natural microcarrier for ex vivo culture andamplification of ESCs in combination with the use of the rotary cell culture system(RCCS). The efficacy of amplification and efficiency of mAM in maintaining theproperties of stem cells were evaluated. mAM loaded with ESC (ESC-mAM) was furthertransplanted to the wound of nude mice to assess the feasibility of using it as a skinsubstitute to repair full-thickness skin defects.2. Methods2.1Obtainment of AM and preparation of mAMUpon approval from the Ethics Committee of the hospital and obtainment of informedconsent from the donors, placentas tissues were obtained within24h of elective cesareansection and all donors were serologically negative for HIV, HBV, HCV and syphilis. AMwas separated from the underlying chorion by blunt dissection.Amniotic cells were removed by repetitive freeze-thawing clycles and DNAenzymatic digestion. AM patches were homogenized into microparticles with amacrohomogenizer in liquid nitrogen to produce microfractures rather than shredding theultrastructure, freeze-dried under a closed vacuum condition, and finally filtrated through ametal mesh filter to obtain300-600μm microparticles.2.2Histological observation of mAMAM samples were routinely fixed and dehydrated, paraffin-embedded, sliced intosections (5μm), and HE stained for observation. Parts of the samples were surface HEstained directly without slicing. The remaining part of the mAM samples were observedunder a field emission scanning electron microscope (SEM) and a transmission electronmicroscope (TEM). The basement membrane structure was observed byImmunohistochemical. 2.3Detection of the growth factors and DNAThe content of the six growth factors (EGF, bFGF, H ELISA GF, TGF-β1, NGF andKGF) in the mAM were assayed by Western blot and ELISA. Total DNA was extractedusing the DNA assay kit and measured with a UV spectrophotometer. Residual DNA onthe surface of mAM was stained with DNA fluorescence and observed under afluorescence microscope.2.4Detection of histocompatibilityThe histocompatibility was evaluated by transplantation of mAM subcutaneously onSD rats.The specimens were retrieved regularly at day3,7,14,30and90(n=5),paraffin-embedded, HE stained, and Immunohistochemical observation of inflammatoryreaction and vascularization. Observation of degradation of AM collagen matrix was usingMasson's trichrome staining.2.5Amplification of ESCs by mAMESCs were isolated by routine "enzymatic digestion and Col IV rapid adhesion"technique and cultured by mAM. The sample was harvested regularly and stained withHoechst33342, and observed under a fluorescence microscope for adhesion andproliferation of the ESCs.2.6Detection of the proliferation and properties markers of ESCsThe efficacy of amplification and efficiency of mAM in maintaining the properties ofstem cells were evaluated. Cell proliferation was measured with the CCK-8and theexpression of β1integrin, CK19, P63and CK10in ESCs was detected byimmunohistochemistry.2.7Construction and Amplification of skin substitute ESC-mAM.The mAM was used as dermal scaffold to construct skin substitute containing ESCs incombination with the use of the rotary cell culture system (RCCS). Addition of new mAMto RCCS was used to amplify the composite skin by bead-to-bead inoculation techniqueautomatically. The specimens were then sliced, HE stained, and observed under a SEM.2.8Transplantation of ESC-mAM to full-thickness skin defectsThe full-thickness skin defect of nude mice was used as animal model for transplantation of ESC-mAM. The efficacy ESC-mAM as a skin substitute to repairfull-thickness skin defects was evaluated. The rate of wound healing was compared andhistological observation was performed regularly.2.9Statistical analysis.All data were analyzed with SPSS16.0and expressed as mean±SD. Pairedcomparisons were performed by Student's t-test, and multiple group comparisons wereperformed by analysis of variance. P<0.05was considered statistically significant.3. Results3.1Physical properties of mAM and DNA contentThe result of repeated thawing and decellularization by DNase digestion showed thateffective cell lysis could be achieved by three thawing cycles. The mAM looked white,semi-transparent and shaped hexahedrally and irregularly. The diameter was300-600μm asmeasured by SEM. HE staining showed that the surface of the matrix was smooth, flat andfully decellularized without residual cells and cell fragments.Hoechst staining showed that mAM nucleic acid was depleted completely from theAM sample. DNA quantitative assay showed that the clearance rate was85±4.15%(13.2±1.3ug/ml versus2.1±0.4ug/ml before and after decellularization).3.2Histological structure and the growth factors of mAMHE staining and SEM showed that mAM was decellularized completely withoutresidual cell fragments, and collagen tissues were continuous in a regular arrangement.TEM showed that the basement membrane structure was continuous and intact, andcollagen fibers were intercrossed. Immunohistochemical observation of basementmembrane-related proteins showed that Ln and Col IV in the matrix of mAM distributedcontinuously and ran throughout the basement membrane. Scattered distribution of Col IVwas also seen in the matrix, in addition to integrins α6/β4. These findings were similar toiAM staining.Western blotting and ELISA showed that NGF, HGF, KGF, bFGF, TGF-β1and EGFexisted in both iAM and mAM.3.3Biocompatibility of mAM HE staining and Masson's trichrome staining showed that there was no significantsign of acute inflammation or rejection reaction. At day14, α-SMA and CD31stainingshowed formation of many microvessels and occasional invasion of major vessels. At day30, a large number of fibroblasts were seen growing in the mAM matrix and partial mAMwas degraded. At day90, most mAM collagen was degraded and fused well with thesurrounding tissue, and fibroblasts were seen distributing homogenously withoutsignificant inflammatory reaction.3.4Amplification of ESCs by mAMESCs were seen adhering to the surface of mAM30min after seeding and growing ina3D manner. By day3, relative proliferative cell activity in RCCS was higher than that inthe plate from day3, and reached326±28%and535±47%by day7and14respectively,which were by far higher than those in the conventional plate culture (232±21%,307±32%,P<0.05).Immunohistochemical observation at day7showed high expression of β1integrin,K19and P63in ESCs of ESC-mAM, reaching93±5.78%,94±4.27%and96±6.35%versus91±4.36%,93±5.18%and94±5.65%in the conventional plate culture. No expression ofK10was observed in either method.3.5Construction and Amplification of skin substitute ESC-mAMUsing mAM as dermal scaffold, skin substitute containing ESCs was constructed andcan be amplified through addition of new mAM to RCCS by bead-to-bead inoculationtechnique. At day14, ESCs had formed2-3layers, and cells between mAM began formingloosely adhering masses of small aggregates.3.6Transplantation to full-thickness skin defectsTwo weeks after transplantation, new epidermis formation was seen in the ESC-mAMgroup, while in the mAM group and blank control group, a residual wound was seen in thecenter of the wound; and the rate of wound healing were significantly lower than that ofthe ESC-mAM group. HE staining of the sections at week4after transplantation showedthat the dermal matrix under the healed epidermis was filled with microparticles in theESC-mAM and mAM groups. In addition, formation of epidermal papillae-like structures was seen in the ESC-mAM group, and these structures were similar to the normal skin. Nosuch formation was seen in the other two groups.4. Conclusion1. Amniotic membrane can be decellularized by repetitive freeze-thawing cycles andDNA enzymatic digestion and homogenized into microparticles with a macrohomogenizerin liquid nitrogen. The mAM not only possesses the characteristics of commonmicrocarriers,but retains the basement membrane structure and bioactivities.2. mAM, which can provide the microenvironment similar to the stem cell nichewithin the human body favorable for ex culture and amplification of ESCs, was able toretain the special properties of stem cells and prevent cell differentiation effectively.3. mAM has a good biocompatibility and can be used as dermal scaffold to construct askin substitute for wound repair. It means that using mAM could achieve amplification ofESCs and construction of skin substitute in one-step, which improve the conventionalmethods of construction by two steps: first amplifying ESCs and then seed them to thedermal scaffold.4. Skin substitute containing ESCs can be amplified by addition of new mAM toRCCS, which not only shortens the time of ex vivo construction but avoids possibledamage to cells due to repeated digestions and maintains the proliferative activity of ESCs,thus providing a novel strategy and way of thinking in promoting the transition of the skinsubstitute to clinical use.