Study Of The Role Of ALX Family Genes In Human Embryonic Stem Cells Derived Neural Crest Cells

Background:Human embryonic stem cells(hESCs)are pluripotent stem cells isolated from inner cell mass(ICM)of human blastocyst.With the capacity to differentiate into cell lineages from all three embryonic germ layers,hESCs hold great promise in numerous fields,such as gene therapy,tissue engineering and drug screening.Neural crest cells(NCCs)are a temporary group of multipotent cells uniquely found in the vertebrates’ embryos.During the early embryogenesis,NCCs derive from the neural folds,migrate throughout the body,and then give rise to a diverse cell lineage of three embryonic germ layers in different microenvironments,including sensory neurons,cartilage and bone,melanocytes,adrenal medullary cells and smooth muscle.As the ability to differentiate into craniofacial cartilage,bone,and connective tissue,NCCs could be used as a cell model to explore the process and mechanism of craniofacial development during early embryogenesis in vitro.Alx gene family is a sub-family of the PRD-class homeobox gene families,consisting of ALX1(Cart1),ALX3 and ALX4.The mutation of this gene family is related to frontonasal dysplasia(FND),an outcome of developmental failure with the facial prominences surrounding the primitive mouth.Moreover,varies of research suggested that ALX gene family plays a crucial role in the craniofacial development.However,the specific expression patterns and functions of these genes are remained uncertain currently.Objectives:1.To differentiate NCCs from H9 hESCs(H9-NCCs)and identify their biological characteristics.2.To clarify the the expressions of ALX family genes in H9-NCCs and their derived cells3.To clarify the functions of ALX family genes in H9-NCCs.Methods and results:1.Derivation and characterization of H9-NCCsMethods:H9 hESCs were induced to differentiate into NCCs(H9-NCCs)by simultaneous activation of Wnt pathway and inhibition of Smad pathway.The cell morphologies of H9-NCCs were observed under microscope during differentiation and immunofluorescent staining and Real-time PCR analysis were performed to test the expressions of specific markers of H9 hESCs and NCCs.Differenet differentiation medium were used to differentiate H9-NCCs into peripheral neurons and mesenchymal stem cells(MSCs)respectively.Furthermore,osteogenic and adipogenic differentiations were further performed to confirm the multipotency of H9-NCCs derived MSCs(H9-NCCs-MSCs).Results:H9 hESCs were grown as compact colonies with clean and defined edges.After differentiation for 7 days,a few rhomboidal cells were migrated from the margin of hESCs colonies and reached confulency after 3 to 4 days(denoted as PO H9-NCCs).Results of Real-time PCR analysis showed that the expressions of NCC markers(SOX9,SOX10,P75,HNK-1,AP2 and ZIC1)were significantly higher in P4 H9-NCCs than in H9 hESCs while the expressions of hESCs markers(OCT4 and NANOG)were dramatically reduced in H9-NCCs.In addition,results of immunofluorescent staining showed that H9-NCCs were positive for expression of HNK-1 and P75 proteins.After 14 days of neural induction,H9-NCCs were elongated with the formation of neurite-like structure.Immunofluorescent staining showed that the differentiatied cells highly expressed β-tubulin Ⅲ and peripherin,suggesting that H9-NCCs were capable of differentiating into peripheral neurons.H9-NCCs changed into spindle shape after cultured in MSC differentiation medium for 5 to 7 days.Flow cytometry analysis showed that majority of H9-NCC-MSCs were positive for the expressions of MSCs surface markers(CD90,CD105,CD73 and CD44)but were negative for the hematopoietic stem cells makers(CD 116,CD 19,CD45 and HLA-DR).Moreover,alizarin red staining showed the formation of calcified nodules after osteogenic differentiation of H9-NCC-MSCs for 14 days.Nevertheless,no lipid droplet was detected by Oil Red O staining of H9-NCC-MSCs after adipogenic differentiation for 14 days.2.The expressions of ALX family gene in H9-NCCs and their derived cells Methods:Used Real-time PCR analysis to detect the expressions of ALX family genes in H9 hESCs,H9-NCCs,H9-NCC-MSCs and their differentiated osteoblasts and peripheral neurons.Results:There was no difference on the expressions of ALX family genes in H9 hESCs and H9-NCCs,whereas the expression of ALX1 in H9-NCCs was significantly higher than in H9-NCC-MSCs and their derived osteoblasts.ALX3 and ALX4 showed higher expression in H9-NCCs-MSCs than in H9-NCCs,while ALX3 was highly expressed in H9-NCCs-MSCs specifically but the expression of ALX4 was highly expressed in peripheral neurons,H9-NCC-MSCs and their derived osteoblasts.3.Effects of over-expression of ALX gene family on the H9-NCCsMethods:Lentivirus carrying ALX-family-genes-overexpression plasmids were harvested and then infected H9-NCCs.Cell Counting Kit-8(CCK-8)was used to characterize the proliferation abilities of cells,Annexin V/7-ADD staining and flow cytometry analysis were performed to analyse the apoptosis of H9-NCCs,and transwell assay was used to assess the migration abilities of H9-NCCs.Results:Up-regulation of ALX1,ALX3 and ALX4 could significantly enhance the migration of H9-NCCs.Interestingly,over-expression of ALX1 remarkably enhanced the proliferation ability of H9-NCCs while over-expression of ALX4 inhibited the proliferation but promoted the apoptosis of H9-NCCs significantly.Conclusion:1.H9-NCCs derived from H9 hESCs possessed the biological characteristics of NCCs for being similar to normal NCCs in morphology and specific markers,as well as showing the potential to differentiate into peripheral neurons and MSCs.2.The expression profiles of different ALX genes in stem cells at different development stages are different,suggesting a temporal and spatial regulation of ALX genes expressions in embryonic development.3.ALX gene family members(ALX1,ALX3 and ALX4)showed both overlapping and distinct effects on the biological characteristics of H9-NCCs.Over-expression of ALX1,ALX3 and ALX4 significantly enhanced the migration of H9-NCCs.Moreover,expressions of ALX1 and ALX4 were related to their proliferation but played contrary roles.Over-expression of ALX1 remarkably enhanced the proliferation of H9-NCCs while over-expression of ALX4 inhibited the proliferation and elevated the levels of apoptotic cells in H9-NCCs remarkably.