| Human i PSCs are highly similar to ESCs: they display ESC-like morphology,they express pluripotency markers,the share a similar level of gene expression and epigenetic states,and they possess the capacity to develop into three germ layers in vitro and in vivo.Given these facts,iPSCs could provide a valuable model to study germ cell determination in vitro,but they could also serve as a promising way to differentiate stem cells into diseased cells in order to model disease,screen drugs,and examine the effects of cell therapy.To date,human i PSCs could have been generated from multiple donor sources,such as skin,adipose tissue,and peripheral blood.However,these cells are obtained via invasive procedures.In contrast,several groups of researchers have found that urine may be a better source for producing i PSCs from normal individuals or patients.This review discusses urinary i PSC(UiPSC)as a candidate for modeling rare diseases.Cells obtained from urine have overwhelming advantages compared to other donor sources since they are safely,affordably,and frequently obtained and they are readily obtained from patients.Thus,Ui PSCs may prove to be a key means of modeling rare diseases and they may facilitate the treatment of those diseases in the future.Objective:The purpose of this research subject is to establish an experiment platform based on integration-free UiPSCs technology,this technology may offer a novel strategy for modeling rare disease.In addition,we performed microarray gene expression profile analysis to compare the differences between UiPSCs and ESCs.A better understanding of their difference is critical for elucidating the reprogramming process and biological characteristics of UiPSCs,for the applications of research and clinical trials using UiPSCs instead of ESCs.Methods:(1)The study utilized the urine from normal individuals to isolate urinary cells(UCs),then made a primary research on the influence factors of the successful isolation of UCs.(2)UCs were reprogrammed to Ui PSCs using episomal vectors that expressed OCT4,SOX2,KLF4 and SV40 LT without using virus,serum,feeder,or oncogene c-MYC.The use of micro RNA clusters 302-367 enhanced the reprogramming efficiency.(3)Alkaline phosphatase staining and flow cytometry were performed to identify and characterize iPSCs.(4)Gene chip was used to compare the gene expression profiles between ESCs and Ui PSCs.Gene ontology annotation,pathway enrichment and gene co-expression network analyses further revealed possible unique functions of these two cell populations.Thus,we selected differentially genes related to reprogramming and differentiation.Then we performed quantitative real-time PCR(qRT-PCR)to detect the expression levels of targeted genes.Results:(1)UCs could be obtained from multiple donors.The isolation of UCs could represent an excellent candidate for noninvasive reprogramming.Besides,to improve the survival rate of UCs,urine should not be collected during the first micturition.(2)We successfully reprogrammed human UCs without using virus,serum,feeder or oncogene c-MYC.(3)AP staining showed differences in reprogramming efficiency between different batches of UCs from the same individual or UCs from different individuals.UiPSCs expressed pluripotent markers including OCT4,TRA-1-60,TRA-1-81,SSEA-4 by flow cytometry.We finished primary identification and characterization of Ui PSCs.(4)We compared the gene expression profiles between ESCs and UiPSCs using bioinformatics analysis.Although the global transcriptional profiles of human ESCs and UiPSCs were globally similar,there was still 19 mRNAs with largest differences in each of the two cell populations.We identified four targeted genes associated with stemness and differentiation including TAF9 B,NNAT,EGR1,PIWIL2 as differentially expressed by q RT-PCR.NNAT,EGR1 were more decreased in UiPSCs than ESCs,which could promote the generation and maintenance of UiPSCs.We also found that a neuron differentiation regulator TAF9 B as the top candidate gene increases in UiPSCs than ESCs(more than 9 fold).Therefore,our result may indicate that the level of TAF9 B in UiPSCs would account for its preference towards neuron differentiation.Conclusions: This study established an experiment method based on integration-free UiPSCs technology without using virus,serum,feeder or oncogene c-MYC,which may open new avenues for modeling rare disorders and offer proof of principle for basic biological research.In addition,we performed a comparative study on the gene expression profile between Ui PSCs and ESCs using microarray technology.Our above results further revealed that UiPSCs and ESCs had different gene expression profiles,in particular in stem property maintain mechanisms.And the high basic level of TAF9 B may partly account for potential of Ui PSCs in neuron differentiation.The molecular differences between Ui PSCs and ESCs described here should drive intense efforts in the future aimed at uncovering UiPSCs as a potential tool in disease modeling,drug discovery and regenerative medicine. |
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