Study On Inductive Differentiation From The Human Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) To The Human Sweat Gland Cells (h-SGCs) And Their Transplantation On Nude Mice

Objective:The human umbilical cord mesenchymal stem cells (UC-MSCs) have thepotential to differentiate to a variety of mature cells, such as chondrocytes,osteoblasts, nerve cells and adipocytes and the procedure for cell separationand culture is relatively easy. UC-MSCs can be obtained through variousresources with high purity, multiplied in vitro amplification times and lowerrisk on allograft immune rejection. Due to their above-mentioned advantages,the usage of UC-MSC has arouse the interest of researchers, particularly interms of tissue repair, in which they can be used for the repair andreconstruction of organs and tissues. This experiment aims to derive theoptimal culture conditions for UC-MSCs by means of studying on theUC-MSCs cell separation through their culture, induction and identificationunder various conditions. Inductive differentiation from UC-MSCs to sweatgland cells through indirect co-culture has also been achieved. The induced,BrdU calibrated UC-MSCs is then transplanted into the nude mice skin andthe observation of their survival and distribution in the mice.Methods:1Separation, culture and identification of UC-MSCs: cut the sterilized,healthy full-term cesarean fetal umbilical cord into umbilical cord tissuesegment with length of3-4cm and washing repeatedly with saline to removethe residual blood. Remove with tweezers the two umbilical arteries and oneumbilical vein after cutting the umbilical cord open to avoid contaminationby the vascular endothelial cells. Tear down the Wharton’s Jelly from theumbilical cord in the shape of streak and scissors them into Wharton’s Jellytissue blocks by1mm3and then place them in culture flask. Use0.25% trypsin digestion to adhere to the cells for passaging when the cells grow fullof bottom. Flow cytometry is used to detect the surface antigen expression incells being cultured.2Drawing of affecting growth factors growth curve of UC-MSCs: UseMTT assay to detect the mean (the OD value) of different concentrationvalues of fetal bovine serum at490nm absorbance values. Use MTT assay todetect the mean (the OD value) of at490nm absorbance values when theplanting density varies. Observing the growth of UC-MSCs in differentperiods after passage and draw the growth curve.3Culture and identification of the human sweat gland cells (h-SGCs):Cut the normal full-thickness skin into organizations blocks by1mm3afterthe removal of subcutaneous fat; obtain the gland tissues by means ofcollagenase digestion Ⅱ and culturing the sweat gland cells (h-SGCs)adherent growth; change the culture medium when most of the sweat glandsclumps become adherent with the following medium changing with theinterval of3-4days. Process the cells being cultured byimmunohistochemistry and detecting the CEA and CK7surface antigenexpression.4Induced differentiation and identification of UC-MSCs: Use47℃water bathing to the sweat gland cells water to render them with in vitro heatshock and use pass three generations (p3) UC-MSCs to indirectlyco-culturing UC-MSCs in order to induce them to differentiate into the stemcells with apocrine phenotype and then processing them byimmunohistochemistry and detecting the CEA and CK7surface antigenexpression.5Making karyotype analysis of the induced stem cells: Join the inducedcells with one drop of colchicine with concentration of0.2mg/L and processthem with hypotonic fixed digest to implement chromosome detection.6BrdU transfection and identification: Apply BrdU antigen of40μg/mlto the passage3(p3) UC-MSCs in order to mark the medium and ifUC-MSCs grow well after48hrs with few suspension cells, measure the transfection by immunohistochemistry assay using stretched preparation.7transplanting the inductively differentiated UC-MSCs in nude mice:process the inductively differentiated UC-MSCs with BrdU calibration andcover them on the defect in the skin wound of the mice along with the humanacellular dermal so as to cover allogeneic skin graft of the mice and thenfix by packaging. Remove the package and the suture after two weeks andmake the biopsy after a month in order to carry out the HE andimmunohistochemical detection. Continuous observation of mice3monthsafter transplantation. Continuously observe the mice for3months aftertransplantation.Results:1Separation, culture and identification of UC-MSCs: fibroblast-like cellshave climbed out around a few umbilical cord tissue blocks after the culture of3-5days. A week later, fibroblast-like cells have climbed out around thevisible part of the tissue block and the medium change has been implemented.The interval for change is2-3days. Passage1stand2ndgeneration (P1, P2) ofthe UC-MSCs elongate bobbin row with morphological uniformity. Thepassage8th(P8) UC-MSCs are still alive, but slow in growth, and the shapechanges and the refraction becomes poor. Some cells have begun to take offthe wall apoptosis. Measuring results of separately cultured UC-MSCs byflow cytometry is that the stem cell surface markers CD29, CD44, CD105have higher expression rate; hematopoietic stem cell surface markers CD14,CD34, CD45almost have no expression, which indicates the cells cultured inthis experiment are relatively purified MSCs.2Drawing of affecting growth factors growth curve of UC-MSCs: MTTcolorimetric assay OD490value tops among the results when theconcentration of the fetal bovine serum is10%with high cell activity andmore living cells. The cell growth curve drawn includes retention period,growth logarithmic phase and plateau.3Culture and identification of the human sweat gland cells (h-SGCs):After3days of culture, there were adherent sweat glands and some cells in irregular shapes had climbed out around sweat glands clumps in the manner ofcolony-like growth; after7days of culture, most of the sweat glandsagglomerates had become adherent and the medium was then changed;12days later, the sweat glands clumps had been surrounded by irregular cells in amanner of cobblestone-like growth.4Induced differentiation and identification of UC-MSCs: process theinductively differentiated cells with immunohistochemistry for the detectionof cell-specific antigen, with result being the pack slurry staining of CEA andCK7are in reddish brown, which indicates to be positive.5Making karyotype analysis of the induced stem cells:30cells wererandomly-chosen for microscope observation and chromosome numbercounting, the result being all chromosome numbers are46, showing nochromosomal aberrations.6BrdU transfection and identification: Immunohistochemical detectionof the visible part of the nucleus indicate they were in brownish yellow.7transplanting the inductively differentiated UC-MSCs in nude mice:through HE staining, cells in the AlloDerm allogeneic dermal were evenlydistributed. Through BrdU antigen expression by immunohistochemicaldetection, the induced stem cells with BrdU calibration were evenlydistributed in the human acellular dermal.Conclusion:1By flow cytometry, the stem cell surface markers CD29, CD44, CD105have higher expression rate and hematopoietic stem cell surface markersCD14, CD34, CD45almost have no expression, which indicates the cellscultured in this experiment are relatively purified MSCs and are capable forexperimental research.2From the experiment, we found that the most suitable environment forUC-MSCs growth is the medium containing10%fetal calf serum and90%DMEM (L), and the planting density that most suitable for the growth ofUC-MSCs is106/ml. UC-MSCs growth rate presents an S-shaped curve withbeginning and end being slow and middle being fast. Through the experiment we can find UC-MSCs within P3are in stable form, high adaptability andstrong metabolism. So they can be used for follow-up experimental study.3It has been confirmed the in the immunohistochemical detection for theinductive of stem cell, both CEA and CK7surface antigen are positive,indicating the induced stem cells have the phenotype of the sweat gland cells.4The chromosomal test results show this indirect co-culture inductivemethod significantly changes the karyotype, suggesting it is a safe and reliableway for induction.5After testing, BrdU transfection is successful.6Animal experiments confirmed the inductively differentiated humanumbilical cord mesenchymal stem cell is safe and effective for transplantationin the skin defects of the nude mice. Continuous observation for3monthsafter transplantation, and no adverse reactions have been found.