Scalable Manufacture Technology Of Neural Stem Cells And Milk-derived Exosomes And Related Translational Medicine Research

Exosomes（Exo）are a population of biologically active extracellular vesicles（EVs）secreted by virtually all cell types.Exo carry the molecular biological information of donor cells and are involved in cell communication and physiological and pathological regulatory processes.It has the application prospect of drug therapy,disease diagnosis,and drug delivery in the clinical study.However,large-scale production of Exo is an obstacle to clinical application.On the other hand,the biological function of Exo is affected by the type of donor cells.Exo derived from different donor cells have different potential for clinical application.In terms of Exo as therapeutic agents,neural stem cell-derived exosomes（NSC-Exo）have unique advantages for treating various neurological diseases.NSC-Exo contain a variety of specific proteins,lipids,and nucleic acid,which are associated with neural stem cells（NSCs）.On the other hand,Exo can avoid the risk of tumorigenicity in cell therapy because it cannot proliferate and spread.However,two of the biggest challenges in obtaining a scalable culture of NSCs for high-efficiency Exo production are the heterogeneity of human induced pluripotent stem cells-derived NSCs（iNSCs）culture and the intrinsic propensity of neuronal or astroglial differentiation.In terms of Exo as drug delivery systems,milk-derived exosomes（milk-Exo）have the properties of abundant sources,low immunogenicity,and resistance to digestions in the gastrointestinal tract following penetrating the intestinal barrier to reach the blood circulation.Compared to exosomes derived from cell culture supernatant,milk-Exo is therefore a natural candidate for drug oral delivery systems for therapeutics,especially for treating chronic diseases that require repeated administration,such as type 2 diabetes milletus（T2DM）.Scalable production of pharmaceutical-grade milk-Exo is more difficult than Exo production from cell culture supernatant because of the abundance of milk dairy proteins and fat.On the other hand,it is difficult to delivery macromolecular drugs such as liraglutide（LRT）into Exo by electroporation due to the lack of intrinsic electrostatic driving force.Thus,to meet the urgent clinical needs for the treatment of nervous system diseases and T2DM,this research proposes potential solutions to the respective challenges and common difficulties of iNSC-Exo and milk-Exo in clinical applications.The research content of this topic is divided into two parts:（1）hiPSCs were used as donor cells for NSCs.TLX,an orphan nuclear receptor protein that plays an important role in regulating proliferation and stemness of NSCs,was over-expressed in hiPSCs,and then hiPSCs^TLXwere differentiated into iNSCs^TLX.TLX,served as an intrinsic signaling regulator for the stemness maintenance and proliferation of NSCs,maintains stability of stemness of iNSCs^TLX,and the problem of long-term stable proliferation of iNSCs was solved by TLX.In addition,a scalable method for isolating and purifying iNSC^TLX-Exo was also developed,providing insight into its therapeutic potential in neurological diseases.（2）The scalable production process of iNSC^TLX-Exo described above was extended to the production of milk-Exo,and then liraglutide（LRT）-loaded milk-Exo（LRT-Exo）was produced.The study of pharmacokinetics and pharmacodynamics was conducted to establish the treatment of T2DM with LRT-Exo by oral administration.Methods and results of first study:（1）Obtaining hiPSCs over-expressing TLX full-length protein（TLX-FL,1-385 aa）or TLX truncated protein（TLX-TP,182-385 aa）by lentiviral infection.The cell line was constructed successfully by identification using quantitative real-time PCR（qPCR）,RNA-Sequencing（RNA-Seq）,flow cytometry（FCM）,and western blot（WB）.（2）Using FCM,RNA-Seq,qPCR,WB,and immunofluorescence（IF）methods,it was demonstrated that TLX could be an intrinsic driving force to regulate SMAD and Wnt signaling pathways for self-drive differentiation of hiPSCs into iNSCs,shorten the differentiation cycle of iNSCs from 14 or 21 days to 6 days,and maintain iNSCs^TLX-FL/TPwith Sox2⁺/Nestin⁺and Vimentin⁺/Musashi-1⁺rates of more than 90%.（3）Using RNA-Seq,FCM,qPCR,WB,karyotype identification,electrophysiology,and IF methods,it was shown that the regulatory ability of TLX-TP on promoting proliferation and maintaining the stemness of iNSCs cells was significantly better than that of TLX-FL,and that iNSCs^TLX-TPcells could remain stable at an expansion rate of 1:15 for at least 45 passages,while still having the potential to differentiate into motor neurons and dopaminergic neurons.（4）Established scalable exosomes purification method based on tangential flow ultrafiltration（TFF）combined with multi-modal chromatography（MM）and density gradient ultracentrifugation（DGUC）.Nano flow cytometry（nanoFCM）,WB,transmission electron microscopy（TEM）,quantitative proteomic analysis,RNA-seq,a rat primary glial cell inflammation model and a rat primary glial-neuronal cell co-culture model were used to characterize and evaluate NSC-Exo.The results showed that the purity of iNSC^TLX-Exo was nearly 90%,and the inflammatory response of rat primary glial cells was significantly inhibited,followed by a significant protective effect on rat primary neuronal cells.Methods and results of second study:（5）The 1 M Na₃PO₄-induced precipitation milk was processed with the purification procedure described above to obtain milk-Exo,and milk-Exo was characterized by nanoFCM,size exclusion chromatography-high performance liquid chromatography（SEC-HPLC）,WB,and TEM.The results showed that the purity of milk-Exo was up to 97% and（1.37±0.55）×10¹⁴particles per 10 L of milk could be obtained.（6）Sublingual,oral gavage,and enteric-coated capsules administration were tested,respectively.The concentration of PKH67-labeled milk-Exo in rodent blood was determined by nanoFCM and normalized to the maximum plasma concentration after tail vein administration to determine the relative bioavailability of each oral route of administration.The results showed that the relative bioavailability of the sublingual route of administration（10%） was significantly higher than that of the other routes of administration（oral gavage:0.6%,enteric-coated capsules:0%）.（7）Peptide drug loading into milk-Exo was achieved by ultrasonic and co-incubation methods.The drug loading efficiency was（6.013±0.163）ng per 1×10⁸particles Exo,which was detected by enzyme-linked immunosorbent assay（ELISA）.Moreover,luciferas was produced by luciferase-GLP-1R-CHO cells after stimulated by LRT-loaded-milk-Exo（LRT-Exo）,which shown that cellular activity of LRT was not affected by ultrasonic.（8）The pharmacokinetics and pharmacodynamics of LRT-Exo were analyzed in db/db diabetic mice.In db/db diabetic mice,sublingual administration of 6×10¹³ particles/kg LRT-Exo resulted in a significant reduction in blood glucose levels comparable to administration of 6×10¹² particles/kg LRT-Exo by tail intravenous injection.The blood concentration of LRT can be maintained stable for at least 24 hours in mice.Conclusions:（1）A method for scalable purification of exosomes based on tangential flow ultrafiltration combined with multi-modal chromatography and density gradient centrifugation has been developed.This method features high purification efficiency,high yield,high sample purity and good repeatability,and is suitable for purification of exosomes derived from both cells supernatant and milk.（2）A population with low heterogeneity and long-term stable passage of iNSCs were obtained by over-expressing TLX in hiPSCs,and the problem of donor cells source for large-scale production of NSC-Exo was solved.（3）The hurdle of loading milk-Exo with peptide drugs was overcome.The oral bioavailability of LRT-Exo was significantly improved by sublingual administration.Blood glucose levels of db/db diabetic mice were also dramatically reduced after sublingual administration of LRT-Exo,which accelerated the transformation of milk-Exo into an orally administrable peptide drug delivery system for clinical application.