Development Of The In-Vitro Culture Systems For Key Cells Responsible For Sika Deer (Cervus Nippon) Antler And Hair Follicle Regeneration And Transcriptome Analysis Of Regeneration-Related Genes

There are large differences in organ regeneration ability among different species.Most mammalian organs lose the ability to fully regenerate after they mature,and can only repair partial cells and tissues.This results in a variety of irreversible degenerative diseases and pain suffering after trauma and aging.Despite the great differences of organ regeneration ability exist in different species and different types of organs,there are often some similarities for different regeneration processes.Therefore,the comparison of different organ regeneration process is a hot research topic and frontier issue in academia during recent years.However,the existing model organisms for organ regeneration are mostly animals with a low evolution level such as salamanders and zebrafish,and they are generally not capable to regenerate multiple organs at the same time.As a result,current research on organ regeneration is limited to lower animals and cross-species organs.This causes interference of evolutionary and interspecies differences for revealing mammalian organ regeneration mechanisms.The vast majority of male deer species of the Cervidea family are characterized by seasonal molting and annual antler regeneration,being capable to naturally achieve periodic complete regeneration of the bone accessory organ-antler,and the mini organ-hair follicle.They are extremely rare mammal with the ability to fully regenerate two types of organs at the same time.This offers a unique opportunity to compare and study the regeneration process of different organs within the same mammal.Therefore,in this thesis,the northeast sika deer(Cervus nippon hortulorum)is selected as the representative of the deer organ regeneration model,and the in vitro culture system for the key cells involved in the regeneration of deer antler and hair follicles is established.Comprehensive comparison and analysis of cell morphology,biological function and gene expression are carried out for these cells and the results are listed as follows:1.We established the in vitro culture system for dermal papilla cells(DerPC)of sika deer.Based on this,two fixed growth patterns of DerPC were constructed: a monolayer 2-dimensional(2D)growth pattern without hair inducing ability and a spherical 3-dimensional(3D)growth pattern with hair inducing ability.These two growth patterns can interchange depending on the culture conditions.DerPC expressed mesenchyme stem cell(MSC)markers and nestin protein.3D DerPC had decreased cell proliferation rate but restored hair regeneration capacity,compared to 2D DerPC.The aggregating dermal papilla cells(MDP)and the aggregated dermal papilla cells(ADP)showed similar gene-expression pattern,while the unaggregated dermal papilla cells(UDP)with 2D morphology showed relatively independent gene-expression pattern.The 317 up-regulated differentially expressed genes(DEGs)in ADP were mainly related to cell adhesion and extracellular matrix(ECM)metabolism.The ECM metabolism-related genes were mainly from the collagen and MMPs families,including COL7A1,COL4A5 and MMP9,etc.The up-regulated expression of ECM-related genes could be an important factor that caused DerPC to restore regeneration ability.The co-expressed 11 DEGs were found in UDP,MDP and ADP,including the ALPL.The up-regulated ALPL during ADP formation may play an important role in the restoration of hair generative capability.2.We established an in vitro culture system for pedicle periosteal cells,including the potentiated pedicle periosteal cells(PPC)and the dormant pedicle periosteal cells(DPC).PPC and DPC showed no obvious differences in morphological structure,proliferation efficiency,stem cell colony number and diameter,expression of MSC markers and nestin,or DEG amounts.After switching to MSC medium,both DPC and PPC could spontaneously aggregate and finally formed 3D spheroids,with no significant difference in the diameter or number of the spheroids.PPC and DPC had3007 co-expressed genes,which were mainly enriched in RNA splicing,transcriptional regulation and post-transcriptional modification,PI3K-Akt signaling pathway,and Ras-MAPK signaling pathway.3.We established an in vitro culture system for antler periosteal cells(AnPC).The proliferation rate of AnPC was significantly lower than those of PPC and DPC.After successive passages,the proliferation rate of AnPC decreased significantly.In addition,AnPC also expressed MSC markers and nestin protein and were capable of forming the stem cell colonies,but the staining intensity,positive staining rate of MSC markers and the number and diameter of colonies were significantly lower than those of PPC and DPC.After switching to MSC medium,AnPC could also spontaneously aggregate and finally form 3D spheroids with increasing stemness,but the number and diameter of the spheroids were significantly lower than those of PPC and DPC.Transcriptome analysis showed that AnPC shared 1270 DEGs with DPC and PPC,including 726 upregulated genes and 544 down-regulated genes.The up-regulated genes in AnPC were mainly related to promoting vascular growth(including VEGFA,NRP1,FOXC2,etc.),neurodevelopment(including WNT5 A,WNT2,SOX11,etc.)and intramembranous osteogenesis(including USP34,MACF1,WNT16,etc.).Our results indicate that the decrease in the regeneration ability of the AnPC compared with PPC and DPC could be due to the decrease in the proliferation rate and stemness of AnPC.On the other hand,AnPC may play an important role in the rapid growth of the regenerated antler by promoting blood vessel growth,nerve development and intramembranous osteogenesis.4.The transcriptome analysis of DerPC,PPC and AnPC suggested PPC and AnPC showed similar gene-expression pattern,while the gene-expression pattern of DerPC are relatively independent.There were 1490 co-expressed genes in DerPC,PPC and AnPC,mainly associated with gene functional clusters related to RNA metabolism,cell cycle,and protein transfer.The interaction analysis showed that the interacting coexpressed genes were mainly related to RNA binding regulation,formation of cytoplasmic translation initiation complex,and proteasome.Finally,three core interacting genes were selected,including UBC,RELA and PSMD4.UBC was predicted to be mainly expressed in the cytosol,nucleus,endosome,endoplasmic reticulum,mitochondria,extracellular and plasma membrane.RELA and PSMD4 were predicted to be mainly expressed in the cytosol and nucleus.The 1565 up-regulated genes in PPC and AnPC compared with DerPC,mainly enriched in the PI3K-Aktm TOR-signaling pathway,calcium ion regulation and immune response gene functional clusters.The up-regulated interaction genes in PPC and AnPC were mainly enriched in calcium ion regulation,activation and response of immune cells,osteoclast differentiation,and Notch signaling pathway.In the end,18 core interaction genes were selected,including CFP,LCK,FYN,etc.The 958 up-regulated DEGs in DerPC,compared with PPC and AnPC,were mainly enriched in guiding the development of tubular structures,branching morphogenesis of an epithelial tube and insulin signaling pathway.The up-regulated interaction genes in DerPC were mainly enriched in morphogenesis of a branching structure,branching morphogenesis of epithelial tube,and focal adhesion.In the end,four core interacting genes were obtained,including ITGB1,LAMA1,ITGA1 and THBS1.Our results indicate that DerPC,PPC and AnPC co-express genes related to the transport and metabolism of RNA and protein that maintain stem cell self-renewal and pluripotency.DerPC specifically expresses a large number of genes that guide hair follicle regeneration and the formation of typical morphological structures of hair follicles,while PPC and AnPC specifically express a large number of genes enriching in osteogenesis and immune regulation that relates to bone regeneration.In summary,we established an in vitro culture system for the key cells(DerPC,PPC and AnPC)involved in the regeneration of hair follicles and deer antlers,and performed a comprehensive analysis of their morphology,biological functions and transcriptomes in this thesis.We found that these cells had certain intrinsic similarities.For example,they had the same fibroblast like morphology,expressed classical MSC markers,could spontaneously aggregate and co-expressed a large number of genes that maintained pluripotency and self-renewal of stem cells.The expression of DEGs causes DerPC and two kinds of periosteal cells to participate in different organ regeneration.These findings provide valuable data for future research of mammalian organ regeneration based on adult stem cells.The established in vitro culture system provides sufficient cell seed resources for studying mammalian trans-organ regeneration.