Effect Of Human Umbilical Cord Mesenchymal Stem Cells On The Proliferation Of Keratinocytes In Co-culture System

Research Background and PurposeAcute and chronic wounds,especially the extensive wounds from skin defects after severe burn(traumatic)injuries,could easily lead to shock,infection,organ failure and other complications,and may be fatal.How wounds can be repaired as early as possible has always remained as the most problematic and pressing issue in the clinical treatment of burn(traumatic)injuries.When the wound surface is exposed,the key in saving the patients is to optimize the use of a small amount of autologous skin to rapidly repair the wounds.Tissue-engineered skin and cell therapy brings new hope to this area,and seed cells is a vital component of tissue-engineered skin.Since allogeneic entities and allogeneic cells with a tissue rejection response can only survive temporarily after transplantation,autologous and allogeneic cells without a rejection response become the ideal choice for seed cells and therapeutic cells.Human umbilical cord mesenchymal stem cells(hUC-MSCs)are derived from the Wharton's Jelly tissue in the umbilical cords.Compared to bone marrow mesenchymal stem cells(BM-MSCs)hUC-MSCs have better self-proliferation,multi-dimensional differentiation,effector cell proliferation functions,and can maintain for a longer time in vitro.In addition,they can be sourced from a wide range of materials in a non-invasive way.They also ethically unrestricted and have a low chance of viral infection.While having immunomodulatory effects,hUC-MSCs can also avoid immunotoxicity from T cells and NK cells.Allogeneic hUC-MSCs hardly cause any immune rejection response without any immunosuppressants.They are also non oncogenic after transplantation.As a result,hUC-MSCs became a major source of mesenchymal stem cells(MSCs),used as therapeutic cells in wound repair,and also used as seed cells in the construction of tissue-engineered skin,among other studies.However,the rate of induced differentiation from hUC-MSCs to Keratinocytes(KCs)is still low(below 15%)under current technologies,and it takes a long time to facilitate the epithelialization of wounds when they are applied separately.However,if hUC-MSCs are used in combination with KCs,it is possible to rapidly provide the KCs required for wound epithelialization while preserving the properties of hUC-MSCs.KCs are the main constituent cells of the barrier skin epidermis.They account for more than 80%of the epidermal cells and can continuously differentiate and renew,keeping the balance between new cells and the exfoliated keratinocytes while producing keratin during the differentiation.Studies have shown that the patients'autologous KCs suspension can accelerate the healing of burn wounds and some chronic wounds.KCs are also indispensable seed cells required in tissue-engineered skin,and since allogeneic KCs can only survive temporarily,autologous KCs are currently being advocated.However,the primary adult KCs has already been differentiated which leads to them having low proliferative or differentiative ability and short lifespan.They have slow growth,short lifespan,and are prone to differentiation and aging under conventional in vitro culture conditionals,making them inappropriate for clinical applications.Studies confirmed that hUC-MSCs can inhibit the apoptosis of KCs and enhance their proliferation and migration,etc.Hence,the combined application of hUC-MScs and KCs can improve the above-mentioned disadvantages of primary KCs.This study aims to discuss whether human hUC-MSCs and KCs can be applied in combination to the construction of tissue-engineered skin seed cells and cell therapies to facilitate wound healing.The effect of hUC-MSCs on the co-cultured KCs'proliferation and the mechanism behind the effects are studied by direct or indirect co-culturing of isolated cultured human hUC-MScs and human KCs.Thus,it provides a theoretical basis for the combined application of hUC-MSCs and autologous KCs as seed cells in the construction of tissue-engineered skin and as therapeutic cells to promote wound healing.Method1.hUC-MSCs are isolated and cultured from the umbilical cords of healthy full-term new-borns by enzymatic digestion;Morphological characteristics of hUC-MSCs are observed by inverted phase contrast microscopes;MTT method is used to detect different generations of hUC-MSCs proliferation and the growth curves are plotted;The cell surface markers are identified by flow cytometry;Multidirectional differential potential of the hUC-MSCs are tested by chondrogenic experiments.2.Primary KCs are isolated and cultured from adult skin using a two-step enzymatic digestion;the morphological observations are done by phase contract microscopes,and the KCs cell markers CK19 and ?1-integrin are detected via immunofluorescence.3.Direct co-culture system is established by grouping the P3 generation of hUC-MSCs and KCs in the ratios of 1:1,1:2,2:1,with pure hUC-MSCs culture group and pure KC culture group established as control groups;MTT method is used to detect the individual OD values of 24 hours,48 hours,and 72 hours groups;The KCs cell markers CK19 and ?1-integrin are detected via immunofluorescence;The EGF and VEGF contents in the cell culture supernatant are determined by ELISA.4.P3 generation hUC-MSCs 1*105 is seeded at the upper layer of the Transwell cell,and the P3 generation KCs 2*105 is seeded at the lower layer of the Transwell cell to establish indirect co-culturing,with the contrast group being pure KCs culture group;MTT method is used to detect the individual OD values of 24 hours,48 hours,and 72 hours cell groups;The EGF and VEGF contents in the cell culture supernatant are determined by ELISAResults1.The cells isolated and cultured using enzymatic digestion from the umbilical cords of healthy full-term newborns are observed under the microscope to display a long spindle or polygonal shape,and they grow in a swirling manner.Flow cytometry detects the overexpression of the isolated cultured cells to be CD 105(98.3±0.9)%and CD90(97.1±1.5)%,and the underexpression to be CD34(1.1±0.2)%and HLA-DR(0.7±0.5)%;MTT methods,which determine the proliferation of hUC-MSCs and plot the growth curve,confirm that after seeding,each generation of cells has an incubation period of one day before entering the exponential growth period which peaks at day seven;Type ? collagen expression is detected by immunofluorescence staining twenty-one days after chondrogenic induction.2.The KCs isolated and culture from adult skin using a two-step digestion are mainly rounded in shape and have regular morphology.The cells start to join into one continuous confluence,resembling the interlocked paving stones;Immunofluorescence staining shows that the cultured cells have positive expression of CK19 and ?1-integrin.3.Observations under the microscope confirm that hUC-MSCs and KCs can be co-cultured and proliferate when they are directly co-cultured in different ratios.MTT method detects the highest OD value in the group which hUC-MSCs and KCs are in a 1:2 ratio,and the OD values of the three co-cultured groups are significantly higher than that of the pure hUC-MSCs and the pure KCs groups(P<0.05);Immunofluorescence staining shows that the number of cells in the co-cultured group is significantly higher than the pure KCs group;The immunofluorescence expression of CK19 and ?1-integrin in the co-cultured group is also enhanced than the pure KCs group;The culture supernatant of the three co-cultured groups contain significantly higher amount of EGF and VEGF than the pure hUC-MSCs group and the pure KCs group(P<0.05),with the group with cell ratio 1:2 having the highest concentration of EGF and VEGF.4.The MTT method shows that the OD values of hUC-MScs and KCs indirect co-cultured group are higher than that of the pure KCs group 24 hours,48 hours,and 72 hours after co-culturing.There is a significant difference in OD values between the indirect co-cultured group and the pure KCs group 72 hours after co-culturing(P<0.05);ELISA shows significantly higher EGF and VEGF levels in the culture supernatant of the hUC-MSCs and KCs co-cultured group than that of the pure KCs group(P<0.05).Conclusion1.This study successfully isolated,cultured,and characterized hUC-MSCs and human KCs in vitro,providing technical support to the research application of hUC-MSCs and human KCs.2.hUC-MSCs can promote the proliferation of KCs in a direct or indirect co-culture system of KCs and hUC-MSCs.This hints the feasibility of a combined application of hUC-MSCs and KCs in the construction of tissue-engineered skin and in cell therapy to enhance wound healing.3.The ability of hUC-MSCs in promoting the proliferation of KCs in direct or indirect co-culture system might be due to their association to paracrine secretions such as EGF and VEGF.