Study Of Optimized System For Generation Of Integration-Free IPSCs From Human Urine-Derived Cells And IPS Disease Model

iPSCs(induced pluripotent stem cells)technology is a big breakthrough in the area of stem cell biology and regeneration medicine.In 2006,Takahashi and Yamanaka find mouse fibroblast cells can be reprogrammed into ESC(embryonic stem cell)by introducing four factors,Oct4,Sox2,Klf4 and c-Myc,in 2007,Yamanaka's lab and Thomson's lab successfully induce human fibroblast into iPSC cells.iPSCs technology not only overcomes ethical concerns regarding the use of human embryos,but also avoids immune rejection after transplantation,because iPSCs induce from self-adult cells.But the insecurity factors included poor induction efficiency,long induction time,virus vector usage and oncogene c-Myc bring a great challenge for its application.Since Junying Yu and her colleagues use episomal vectors to induction iPSCs free of vector and transgene sequence in 2007,the insecurity of iPSCs is greatly increased,making a major step forward to clinical application.Two year later,they also increase episomal reprogramming efficiency using small molecules.Hongkui Deng group successfully induced mouse adult cells into iPSCs only using the combination of six small molecules in 2013,this significant progress has been widespread concern worldwide,meanwhile,scientists aware also small molecules which isn't transgene can regulate gene expression and decide cell fate by complex molecular mechanisms.IPSCs induced using small molecules or transgenes must perform rigorous evaluation,but human iPSCs induced only by small molecules hasn't been reported.From cell-derived iPSCs,blood cells,skin fibroblasts,amniotic fluid cells,keratinocytes epithelial cells,hair follicle cells and other type cells have been achieved reprogramming,but most of them are invasive cell source,human urine-derived somatic cells(UCs,urine cells)characterized in drawn convenient,non-invasive,no immune rejection and ethical issues,less accumulated mutations are ideal source of iPSCs.But non-integrated reprogramming method has long induction time,poor induction efficiency,low success rate,UCs came from different people have very different proliferative ability and survival in the programming,leading to difficult to large-scale application of this method.A natural fatty acid sodium butyrate(NaB)can greatly improve reprogramming efficiency by promoting epigenetic remodeling and pluripotency genes expression,CHIR99021(Chir)is a GSK-3? inhibitor,when it is used combing with LSD1 inhibitor,human keratinocyte cells could be induced into iPSCs only by introducing two factors OCT4 and KLF4.P53 has been demonstrated to be a barrier of reprogramming,downregulation of P53 improves reprogramming efficiency,and promotes self-renewal of ESCs.Yunying Yu et,al.find a combination of small molecules,including MEK inhibitor PD0325901(PD),GSK-3? inhibitor Chir,TGF-?/Activin/Nodal receptor inhibitor A-83-01,ROCK inhibitor HA-100 and LIF inhibitor,promotes the non-integrated reprogramming and obtains clinical grade iPSCs.In this study,we optimized the reprogramming method based on traditional human urine cell non-integrated reprogramming system,in Part 1,we found a small molecules combination reprogramming iPSCs with high efficiency,including A-83-01,Chir,NaB,Cyclic Pifithrin-a(CPFT-a),Thiazovivin(Tzv)and PD,by screening more than 20 small molecules reported previously.Through the study of small molecules acting time,we found five small molecules suitable for the early reprogramming added,PD which could induce apoptosis of non-iPSCs,was added in the later stage of reprogramming.For UCs with less initial number,poor proliferation,low survival after electroporation,we used autologous UCs as feeder cells to provide survival and proliferation of stromal environment for UCs after electroporation.After the optimization of the above induction system,we construct a set of induction methods for different proliferation ability of UCs:Plan A:RM at day 0?1,RM+5M at day 2?9,mTeSRl at day10?13.UCs were seeded on Matrigel.This method is suitable for UCs with good proliferation ability.Plan B:RM at day 0?1,RM+5M at day 2?9,mTeSR1+6M at day10?17.UCs were seeded on Matrigel.This method is suitable for UCs poor proliferation ability,but could up to 100%confluence at fifth day.Plan C:Medium was the same with Plan B,but seeded on autologous UCs as feeders before nucleofection.This method is suitable for UCs with poor proliferation,low survival after electroporation,but couldn't up to 100%confluence at fifth day.The usage of small molecules combination improved the induction efficiency up to 170 times and the success rate to 100%,and shorted the induction time to 13 days.Further studies showed small molecules combination downregulated 14 known reprogramming barriers,suggesting the small molecules combination blocked p53,HDACs,TGF-?,GSK3?,ROCK and MEK signaling pathway and inhibited a series of obstacle reprogramming genes to increase the reprogramming efficiency.This method also is suitable for human amniotic fluid cells,this method also reprograms human umbilical cord-derived mesenchymal stem cells,but with low efficiency.PFT-a was first used in iPSC technology,compared to p53 shRNA,its dosage and treatment time could be flexibility control.We further investigate the characters of iPSCs,and found the karyotype was correct,alkaline phosphatase staining was positive,OCT4,SSEA4,TRA-1-60,TRA-1-81 and other pluripotency genes were high expression,the expression of exogenous genes of plasmid weren't insert into the genome,the promoters of OCT4 and Nanog weren't methylated,teratoma formation analysis suggested iPSCs could differentiate into three germ layers.Transcriptome of iPSCs was highly similar with ESC transcriptome.This induction method not only greatly improved the reprogramming efficiency,shorting induction time,but also helped the UCs with poor proliferation complete reprogramming,and effectively improve the success rate of proliferation.So that this technology can be widely used preparation of human iPSCs,and conductive to the establishment of human iPSCs bank.In the process of the establishment of human iPSCs bank,we found differences in the efficiency of reprogramming human cells in urine were large,reprogramming efficiency was also very different in different batches of the same person,We wanted to found this difference comes from different people individual heterogeneity or another reason.UCs were isolated from urine,in theory,UCs may come from the department of urine.From the structure of urinary system,it is estimated that each human kidney contains more than 1 million nephrons and processes?100 liters of filtrate per day30.Yet,most of this volume is reabsorbed in the distal part of the nephron and only?2 liters are normally excreted through the urethra as urine daily.Perhaps not surprisingly,given the massive tubular network,2,000-7,000 cells are detached from this system daily and can be collected in urine.Most cells are dead,only a few cells can survival,adherent and amplification.For the tissue-derived cells in the urine,there are many possibilities,such as podocytes,renal cell wall,renal vascular endothelial cells,renal tubular epithelial cells collecting duct cells,cell ureter,bladder epithelial cells and urinary tract cells.Previous reports suggest UCs come from tubular epithelial cells.However,in the process of a large number of extraction and amplification,we found morphology of cells in urine from the same people weren't consistent,suggesting the multiple sources of UCs.In the Part 2,we purified different kinds of UCs using cloning method,and found there were three types of UCs could be successfully cultured,we named them as Type 1,Type 2 and Type 3.When these types UCs were performed reprogrammed,Type 1 and Type 2 UCs could complete reprogramming using our method,but Type 3 UCs couldn't complete reprogramming.This result suggested different types of UCs had different reprogramming ability using the same induction method.We screened the Type 1 and Type 2 UCs before induction to improve the induction efficiency and reduce the costs.Further analysis found Type 1 and Type 2 UCs came from renal tubular epithelial,and Type 3 UCs came from bladder epithelium.This showed that renal tubular epithelial could been reprogramming easily,bladder epithelium couldn't been reprogramming.So far,we have identified tissue source for the reprogramming of UCs,and gained iPSCs derived from single cell with identied.This result not only helped solve the problem of poor reproducibility results of biological samples in basic research,but also helped establish the stable differentiation system of iPSCs and was important for further clinical application.Tubular epithelial-derived iPSCs was an ideal source for kidney induction in vitro,because epigenetic memory of induction cells was identical with iPSCs,making the establishment of high-quality disease models as possible.iPSCs technology has been widespread concerned and studied in-depth by many laboratories,especially terms whose study involved in regenerative medicine and disease models.Establishment of human iPSCs bank can be used to construct diseases specific cell models to study the mechanism.Recent years many groups has made great progress and breakthroughs in the field of neurological diseases,eye diseases,urinary system diseases,endocrine system diseases and so on.The of diabetes is high,the number of Chinese with diabetes accounts for one-quarter of the world diabetes in 2013,more than 80%was type 2 diabetes(T2D)with complex causes,influenced by genetic and environmental factors,1-5%was special types of diabetes,maturity-onset diabetes of the young diabetes(MOD Y)was one of them which belongs to a single gene mutation disease,its etiology is very clear.Knockout mouse or mouse islet cells were used as the model previously,and had a species difference.There is a great difference in the anatomical position in mouse and human pancreas:former,such as dendritic dispersed on duodenal mesentery,which is located between the duodenum and spleen.There are also large differences in islet structure:mouse islets just have one subunit,and human pancreatic islets formed by several subunits.Process development and pathogenesis of pancreatic diabetes and mouse are different,but the human cell model to simulate reported the development process and the pathogenesis of the disease is very small.The gene targeting technology and iPS technology are used in diabetes research,and are advantageous for specific genes or specific mutations of specific genes.Islet ? cell body induced from autologous iPSCs is not limited by the source of supply,and has no immune rejection and ethical issues,it was undoubtedly that it benefited a huge number of people with diabetes.After a single gene repair induction iPSCs differentiate into insulin-secreting cells(insulin producing cell,IPC)will be a major achievement of stem cell biology to clinical transformation.In part 3,we explored the method about islet cell differentiation from iPSCs,and collected UCs from patients with MODY of a single gene mutation.The islet differentiation has three-step and five-step method,from induction environment has induced in vivo and in vitro,from inducing factor has small molecules only and gene expression using viral vectors.We chose two methods:one is simply use small molecules,five-step method,islet cells differentiated from the endocrine progenitor cells.Anthor is a transgenic approach by constructing pMx-PDX1,pMx-Ngn3 and other reverse transcriptase viral vectors,viral packaging and in an appropriate time to infect UCs to induce iPSCs,in turn to achieve the induction of islet.But because of long induction time and unstable induction system,it was difficult to get islet which can secret insulin.We just completed iPSCs induced by small molecules,inwardly endoderm differentiation and the vector construction used in Method 2.We have screened diabetic patients with a family history of diabetes,and visited them to confirm family history of illness of patients and to collect urine or blood,performing genetic diagnosis.We chose MODT with HNF1a,HNF4a and GCK for further analysis using PCR(polymerase chain reaction)and gene sequencing,but we did not find MODY mutation.only found multiple SNP,so we still couldn't confirm with the correlation between diabetes and these mutation.We preliminarily explored the islet differentiation method from iPSCs,and successfully constructed a number of islet differentiation key gene retroviral vector,we have accumulated rich experience in the collection of cases and genetic diagnosis work.This will help establish a successful model in the future to study disease pathogenesis.In summary,the present study improved the induction method for non-integrated iPSCs from UCs,and promoted the induction efficiency of UCs with poor proliferation,it was conducive to build iPSCs bank.We also found the resour of UCs of reprogramming,and got iPSCs form a single cells.We also explored the process of islet differentiation form iPSCs,and accumulated experience for screening single gene disorders.specifically for heavy tissue-derived cells in urine programmed to give a single cell-derived iPSCs identity determined;a preliminary exploration of the process of differentiation of iPSCs to insulin,and looking at the single gene disorders in the process of accumulated experience.