Generation And Identification Of Induced Pluripotent Stem Cells From Cord Blood Of Two Kinds Of Skeletal Dysplasia Fetuses

BackgroundCleidocranial dysplasia(CCD;MIM #119600)is a rare autosomal bone disorder resulting from delayed or abnormal ossification of bony growth,which is caused by heterozygous loss-of-function mutation of the RUNX2 gene.RUNX2 is an important transcription factor for cranial suture closure and membranous bone morphogenesis,which is expressed in multipotent mesenchymal cells,osteoblast-lineage cells,and chondrocytes,and plays a master role in the differentiation of osteoblasts and the maturation of chondrocytesAchondroplasia(ACH;MIM #100800)is a rare autosomal dominant congenital disease representing the most common form of short-limb dwarfism in humans,which is caused by the gain-of-function mutation in the fibroblast growth factor receptor 3(FGFR3)gene.Most patients with ACH have the heterozygous G380 R mutation in FGFR3 gene,which affects bones development predominantly through endochondral ossification.Despite years of research,several aspects of FGFR3 function in ACH remain unclear or controversial due to the lack of ideal in vitro disease model.Induced pluripotent stem cell(iPSC)were generated from various mammal somatic cells through the utilization of reprogramming technology.Owing to Their biological characteristics which are similar to those of embryonic stem cells and it’s facing fewer ethic problems,iPSC have been acted as suitable disease cell models.In this study,PBMC from a CCD fetus with exon 3 heterozygous deletion in RUNX2 gene and an ACH fetus with heterozygous c.1138G>A(p.Gly380Arg)mutation in FGFR3 gene were isolated from the fetal umbilical cord blood sample.And then PBMCs were electroporated with a mixture of episomal plasmids expressing OCT4,SOX2,c-MYC,KLF4 and Bcl-XL.This iPSC lines are valuable in vitro models to study the pathological mechanism and the treatment of CCD and ACH.ObjectivesTo generate and identify two iPSCs line via PBMC from a CCD fetus with exon3 heterozygous deletion in RUNX2 gene and an ACH fetus with heterozygous c.1138G>A(p.Gly380Arg)mutation in FGFR3 gene,which will be acted as suitable disease cell models to study the pathological mechanism and the treatment of CCD and ACH.MethodsWith the approval of pregnant women and their family,fetal umbilical cord blood sample from a CCD fetus with exon 3 heterozygous deletion in RUNX2 gene and an ACH fetus with heterozygous c.1138G>A(p.Gly380Arg)mutation in FGFR3 gene were obtained after termination of pregnancy.And PBMC were isolated from the fetal umbilical cord blood and cultured in PBMC medium.To establish the iPSC,PBMCs were reprogrammed by episomal vectors encoding OCT4,SOX2,c-MYC,KLF4 and BCL-XL.STR analysis,karyotyping and Sanger sequencing were used to identify whether the iPSCs were drived from the original PBMCs.Alkaline phosphatase(AP),immunofluorescence staining,RT-qPCR and in vitro directed differentiation were used to identify the multi-directional differentiation of GZHMCi003-A and GZHMCi004-A.ResultsGZHMCi003-A and GZHMCi004-A exhibited a normal morphology and stained positive for AP.Expression of endogenous pluripotent markers NANOG,OCT4 and SOX2 was detected by RT-qPCR,which was comparable to the positive control H9-h ESC line.Immunofluorescence staining was also performed to confirm the expression of the pluripotent markers SOX2,OCT4,NANOG and TRA-1–60.Karyotype analysis showed that GZHMCi003-A and GZHMCi004-A had a normal diploid karyotype(46,XX).Exon 3 heterozygous deletion in RUNX2 gene was verified in GZHMCi003-A by qPCR,and heterozygous G380R(c.1138 G > A)mutation in FGFR3 gene in GZHMCi004-A were verified by Sanger sequencing,respectively.In vitro directed differentiation was performed to assess the differential potency of GZHMCi003-A and GZHMCi004-A.And both of them could differentiated into all three germ layers,including ectoderm,mesoderm and endoderm.The absence of exogenous reprogramming factors was confirmed by PCR.GZHMCi003-A and GZHMCi004-A were negative for mycoplasma contamination by PCR.Finally,STR analysis showed that the genetic identity of GZHMCi003-A and GZHMCi004-A were the same as the parental PBMC.ConclusionPBMC with CCD and ACH gene mustation can be reprogrammed into patient-derived iPSC.And they will become not only suitable disease cell models to study the pathological mechanism and the treatment of CCD and ACH,but well material to establish the patient-derived organoid of CCD and ACH.