Identification And Characterization Of Key Chemokines That Regulate Human Pluripotent Stem Cells

Aims Microenvironment(or niche)-dependent chemokines play multiple biological functions during the development of early and adult tissue-specific stem cells.However,to what extent chemokines influence human pluripotent stem cells(h PSCs)is not yet completely understood.In this study,we established a new human embryonic stem cell(h ESC)line and human-induced pluripotent stem cell(hi PSC)line.These cells were then used to explore the effects of different culture conditions(KSR and m Te SR1 media)on h PSC maintenance in vitro.In addition,according to the two different conditions,we applied protein array to screen highly expressed chemokines found within the cytokine pool in the culture supernatant of h PSCs.The sources of these chemokines in the supernatant were further identified.Finally,in regard to the role of high chemokine expression in the supernatant on the regulation of migration and pluripotency of h PSCs in vitro,we determined that key chemokines have an important regulatory role in h PSCs.Methods(1)We isolated the inner cell mass(ICM)from blastocyst and established a new h ESC line through immunosurgery.The pluripotency of these h ESCs was then evaluated/identified using G-band karyotyping,alkaline phosphatase(AP)staining,immunofluorescence,and real-time PCR.Moreover,embryoid body(EB)differentiation in vivo and teratoma formation assay in vitro were conducted to support the pluripotency and multiple differentiation potential of h ESCs.(2)We introduced four transcription factors Oct4/Sox2/Klf4/c-Myc into somatic cells(human foreskin fibroblasts,HFFs)through a retrovirus system.The HFFs were induced and reprogrammed into ES-like-induced pluripotent stem cells,with the pluripotency of these new hi PSCs preliminarily tested by immunofluorescence.(3)We used two recognized culture conditions(KSR or m Te SR1 media)for h PSCs to test the maintenance of h ESCs and hi PSCs.(4)Protein arrays were applied to screen chemokines that resided in the cytokine pool within the culture supernatant of h PSCs,and the source of these chemokines was further identified.(5)We performed h PSC transmigration assay to explore the biological functions of the chemokines in h PSCs using three exogenous human recombinant proteins as inducers,that is,IL-8,IP-10,and SDF-1a.We used cognate receptor antagonists(CXCR1/2-specific antagonist reparixin and SB265610,CXCR3-specific antagonist NBI74330,and CXCR4-specific antagonist AMD3100)to block the endogenous signaling pathway and further verified the ligand-induced migration effect.(6)Real-time PCR was used to detect m RNA expression levels of pluripotency genes(Oct4,Nanog,and Rex-1)and differentiation genes(AFP,c-actin,and Sox1)in h PSCs treated with the exogenous chemokines and cognate receptor-specific inhibitors.We determined the roles of the IL-8-CXCR1/2,IP-10-CXCR3,and SDF-1a-CXCR4 signaling pathways in maintaining cultures of h PSCs in vitro.Results(1)We successfully obtained a new h ESC line.The h ESCs exhibited self-renewal,continuous expansion,and normal karyotype,and AP activity was high.These cells also displayed high expression levels of pluripotency genes(Oct4,Nanog,and Rex-1)and pluripotency markers(Oct4,Nanog,TRA-1-81,and TRA-1-60).Furthermore,the h ESC-derived EBs could differentiate into three germ layers(i.e.,endoderm,mesoderm,and ectoderm)and were highly expressed with three germ layer-markers(AFP/GATA4,Desmin/Actin,and Nestin/b-III Tubulin).Finally,the h ESCs were injected intramuscularly into the hind leg muscles of NOD SCID mice,with the integrated germ layer-tissues forming teratomas.(2)We also acquired a new hi PSC line.Similar to the h ESCs,the hi PSCs possessed the capacity of self-renewal and proliferation,and highly expressed the four pluripotency markers.(3)The h ESCs and hi PSCs showed high AP activity and maintained their pluripotency under both KSR and m Te SR1 culture systems;specifically,the four stemness/pluripotency markers were highly expressed in the two cell lines.(4)The relative signal intensity(RSI)of 38 chemokines protein arrays showed that the h ESCs-m Te SR1 and h ESCs+Feeder cells-KSR supernatants contained greater variety and higher concentrations of chemokines.The secretions from h ESCs/hi PSCs and feeder cells were the primary chemokine component of the h PSC culture supernatants.(5)Exogenous recombinant IL-8/IP-10/SDF-1a proteins significantly induced h PSC migration in vitro,and the receptor-specific antagonists effectively blocked the process,suggesting that these chemokines mediated the migration of h PSCs.(6)After the receptor inhibitors reparixin/SB265610 blocked the signals,the expressions of the three pluripotency genes were significantly upregulated and the expressions of the three differentiation genes were significantly downregulated.However,the opposite was true for IP-10 and SDF-1a.These results indicated that the IL-8-CXCR1/2 signaling pathway tended to enhance the differentiation of h PSCs,whereas the IP-10-CXCR3 and SDF-1a-CXCR4 signals tended to enhance the pluripotency of h PSCs.Conclusions This study demonstrated that chemokines were the predominant signal proteins,and that IL-8,SDF-1?,and IP-10 were important chemokines that mediated the migration of h PSCs and h ESCs.These results support that chemokines secreted from both stem and feeder cells are essential for mobilizing h PSCs and maintaining their stemness.