Using Co-culture System To Reveal The Regulation Role Of Macrophages To Antler Stem Cells

Organ damage or functional decline currently is one of the major health problems faced by human beings.More than 4 million people suffer from defects or dysfunction of tissues or organs every year due to trauma or disease.Currently,transplanting new organs is the main means to deal with the problem.Although immunosuppressive drugs and transplantation immunology have made great progress,the sources of organs available for transplantation are limited,and immune rejection is prone to occur.To solve the problem fundamentally,the key is to stimulate regeneration of the defective organs de novo.Velvet antler is the only mammalian organ found to be fully regenerable,and is an ideal model for studying the complete regeneration of mammalian organs in nature.Recent studies have found that the initiation of regeneration is closely related to the regulation of the immune system,but the regulation mechanism of the immune system in the regeneration of antler has not been reported thus far.Antler regeneration is a stem cell-based regeneration process.The pedicle periosteum cells(PPC)are the key cell types for antler regeneration.The PPCs could be induced to differentiate into adipocytes and chondrocytes.Fluorescence assay was used to examination of the expression of stem cell markers in PPCs.The results showed that the surface markers of mesenchymal stem cells CD9,CD 105,CD73,Stro-1 and neural stem cell marker Nestin were highly expressed in PPCs,and PPCs did not express hematopoietic stem cell markers CD34,and exhibited high proliferative activity when cultured in vitro.These findings indicate that the PPCs have the characteristics of stem cells and are a special type of adult stem cells for antler regeneration.In this study,PPCs were cultured in conditioned medium of macrophages,and thereafter sequenced at transcriptome level.The high-quality sequences obtained after quality control were compared with the white-tailed deer genome to obtain mapped data,gene expression level was calculated by FPKM method.The differentially expressed genes(DEPs)were detected by using |Fold Change|?2 and FDR<0.01 as the screening standard,and the GO gene enrichment annotation was performed on the DEPs by DAVID software.The results showed that the ratio of bases with a quality of not less than 20(Q20)was higher than 95%after sequencing,and 112 DEPs were screened.Among them,the experimental group had 38 DEPs up-regulated,74 down-regulated.The GO gene enrichment annotation of DEPs in the experimental group was mainly involved in extracellular matrix,chemotaxis,and biological processes,such as wound healing and growth factor-stimulated responses,bone formation,etc.It is speculated that macrophages may alter PPCs growth by secreted cytokines.Therefore,macrophages regulate the physiological state of PPCs maight be through altering their microenvironment,and indirectly participate in the regeneration process of deer antler.