| In Greek mythology,the Titans Prometheus' liver was constantly pecked by the Condor bucause of Zeus' punishment.Amazingly,his liver could constantly regenerate after being pecked,so his life could be maintained.In modern society,patients with organ failure caused by chronics and organ injury caused by accidents are increasing year by year.Faced with these diseases,the existing traditional medical methods often seem powerless.So turning myth into reality is urgently needed.Regenerative medicine is the discipline that studying replacement of damaged cells,tissues or organs with normal ones to cure diseases,and it is an important medical research direction in the future.The shortage of cells,tissues or organs is one of the most important factors limiting the development of regenerative medicine.Human Pluripotent stem cells(hPSCs)can go indefinite self-renewal and hold the potential to give rise to all cell types in the human body,thus could continuously provide various types of cells,tissues and organs for regenerative medicine theoretically and have great prospects.Since the first human pluripotent stem cell line-human embryonic stem cells(hESCs)was established by Thomson laboratory in 1998,scientists have continued to explore the clinical application of hPSCs and have made a series of achievements.Since hESCs are derived from early human embryos,which is related to ethical issues,the basic research and clinical application of hESCs are controversial.Human induced pluripotent stem cells(hiPSCs),which share identical cell characteristics and clinical application values with hESCs,are derived by delivering exogenous genes into somatic cells or treating somatic cells with chemical molecules,so there are no ethical issues involved in hiPSCs.Low reprogramming efficiencies,exogenous genes randomly integration and spontaneously activation,usage of animal-derived reagents,genomic and epigenomic instability and others are major factors that affect clincial application of hiPSCs.With the development of technologies and theories in the basic research of hiPSCs,it is time to pave the way for their clinical applications.Now scientists in Japan are doing a hiPSCs based clinical trial,which uses Retinal Pigmented Epithelial cells(RPE)differentiated from hiPSCs for autologous transplantation to treat age-related macular degeneration[1]Until now,there are no side effects observed in patients who have received cell transplalntation and the patients' eyesights have improved to some extent.To ensure the safety,effectiveness,traceability,reproducibility and legality of hiPSCs intended for clinical trials or therapies,the following principles should be followed:(1)Parental cell donation process must comply with the "Guidelines for Human Somatic Cell Therapies and Quality Control of Cell-based Products" and the "Tissue Donor Guidance".(2)Parental cells must be easy to obtain.(3)In clinical applications,cells from old individuals which are difficult to be reprogrammed will be inevitably involved,so high reprogramming efficiency are required to ensure success rate.(4)To avoid insertional mutations caused by exogenous genes random integration and tumorigenicity caused by exogenous genes spontaneous activation,hiPSCs intended for clinical researches or therapies should be exogenous factors free.(5)According to the current national and international regulation policies,most countries require a Good Manufacturing Practice(GMP)environment and operation procedure when handling the cells for clinical applications.So operation procedures and manufacturing environments of parental cell collection and cultivation,hiPSCs induction,hiPSCs differentiaions should be comply with the current GMP standards.(6)To avoid cross-transmission of animal pathogens between humans and animals,it is better to avoid using ingredients of animal origin in the cell culturing process.(7)Bacteria,fungi,mycoplasma and some types of pathogen must be detected to ensure the biological safety of clinical-grade cells.Previously,one group indicated that hiPSCs could be derived under fully defined conditions from the very beginning,however,whether the obtained iPSC lines were biologically safe or the"tissue donor guidance" was followed were not described.Several other groups have also successfully achieved the clinical-grade hiPSC lines by converting the existing non-clinical-grade hiPSC lines.However,in this process animal products were applied at the beginning,which could cause the risk of infectious agents transfer across-species.So it is urgent to derive clinical-grade hiPSC lines in GMP and Xeno-free conditions following the "tissue donor guidance" from the very beginning and evaluate the safety of the cell lines strictly.Based on the basic requirements and the current progresses in clinical-grade hiPSCs,we conducted the following researches:(1)Firstly,we optimized the reprogramming process from human umbilical cord blood mononuclear cells(hUCBMCs),which are easy to obtain,to hiPSCs,and achieved high reprogramming efficiency and success rate.(2)Secondly,we optimized the process of non-integrated plasmid mediated reprogramming.Without the proto-oncogene C-Myc,we sucessfully reprogrammed human fetal fibroblasts(HFF)into hiPSCs by a non-integrated plasmid carring Oct4?Sox2?Klf4 and Nanog with high efficiency.(3)In the process of optimizing non-integrated plasmid mediated reprogramming,we found that reprogramming efficiencies differs in HFF cells from different individual in an age independent manner.Then we conducted RNA-Seq among these cells and analyzed their gene expression profiles to identify the underlying mechanisms that leading to the different reprogramming efficiencies.(4)Finally,we sucessfully established several integration-free clinical-grade hiPSC lines in GMP and Xeno-free conditions following the "tissue donor guidance" from the very beginning and tested their cell characteristics,differentiation ability and biological safety comprehensively.Our results showed that:(5)Before reprogramming,converting the parental hUCBMCs from suspension culture state into adherent state by coating the Petri dish with MesenCult-XF attachment substrates,and during reprogaming,adding feeder cells to the lossely attached hUCBMCs instead of seeding the hUCBMCs onto feeder cells can greatly improve the reprogramming efficiency and success rate from hUCBMCs to hiPSCs.(6)HFF-21WF cells can be reproammed to hiPSCs by the non-integrated plasmid carrying Klf4 and VP 16 engineered Oct4,Sox2 and Nanog(PCEP4-OSKN)efficiently in the case of using the highly efficient electrotransfection gene transfection method for exogeneous genes delivery and using X medium during the reprogramming process.Our reprogramming efficiency was significantly higher than other episomal mediated oncogene-free reprogramming efficiencies,and hiPSCs lines can be established without the use of small molecules involved in epigenentic regulation.(7)The non-integrated plasmid,PCEP4-OSKN,could reprogram HFF-21WF efficiently.However,it could not fully reprogram UCMSC-21WF and and other HFF cells from different individuals.(8)RNA-Seq results of the four HFF ceels(HFF-21WF,HFF-19WM,HFF-16WM and HFF-6Y)and the human umbilical cord mesenchymal stem cells(UCMSC-21WF)indicate that differentially expressed genes between HFF-21 WF cell line(with high reprogramming efficiency)and other cell lines(with low reprogramming efficiency)are mainly cell junction related genes and TGF? signaling related genes.(9)Integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture could be suceessfully established in line with the current guidance of international and national evaluation criteria.Neural cells and cardiomyocytes could differentiate from the established hiPSCs in Xeno-free culture medium.The established hiPSCs were biologically safe.This study may provide good seed cell source and reprogramming method for future hiPSCsbased clinical trials or therapies. |
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