技术资料

Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1,encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it,we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However,the mALK2 leads to inhibitory pluripotency maintenance,or impaired clonogenic potential after single-cell dissociation as an inevitable step,which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus,current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome,and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors,CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617GtextgreaterA. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern,as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time,labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies,which is hampered by inhibitory pluripotency-maintenance requirements,or vulnerability of single-cell-dissociated iPSCs. View Publication

It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ˜50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor,UBTF,from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency,hESCs were treated with the Pol I inhibitor,CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers,reduces the expression of pluripotency markers,and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene,and corresponding reduction in transcription,represent early regulatory events during the directed differentiation of pluripotent stem cells. View Publication

In most human somatic cells,the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually,DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However,the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped,in contrast to fibroblast cells that enter a state of replicative senescence. Significantly,telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity,we can functionally separate the two unique properties of human pluripotent stem cells,namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation,we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo,and thus sustained telomerase activity. This article is protected by copyright. All rights reserved. View Publication

Current long term cryopreservation of cell stocks routinely requires the use of liquid nitrogen (LN2),because commonly used cryopreservation media containing cell membrane permeating cryoprotectants are thermally unstable when frozen at higher storage temperatures,e.g. -80 °C. This instability leads to ice recrystallization,causing progressive loss of cell viability over time under the storage conditions provided by most laboratory deep freezers. The dependency on LN2 for cell storage significantly increases operational expense and raises issues related to impaired working efficiency and safety. Here we demonstrate that addition of Ficoll 70 to cryoprotectant solutions significantly improves system thermal stability at the working temperature (˜-80 °C) of laboratory deep freezers. Moreover,a medium comprised of Ficoll 70 and dimethyl sulfoxide (DMSO) in presence or absence of fetal bovine serum (FBS) can provide reliable cryopreservation of various kinds of human and porcine pluripotent stem cells at -80 °C for periods that extend up to at least one year,with the post-thaw viability,plating efficiency,and full retention of pluripotent phenotype comparable to that achieved with LN2 storage. These results illustrate the practicability of a promising long-term cryopreservation method that completely eliminates the need for LN2. View Publication

Utilizing a fast 31P magnetic resonance spectroscopy (MRS) 2-dimensional chemical shift imaging (2D-CSI) method,this study examined the heterogeneity of creatine kinase (CK) forward flux rate of hearts with postinfarction left ventricular (LV) remodeling. Immunosuppressed Yorkshire pigs were assigned to 4 groups: 1) A sham-operated normal group (SHAM,n = 6); 2) A 60 minutes distal left anterior descending coronary artery ligation and reperfusion (MI,n = 6); 3) Open patch group; ligation injury plus open fibrin patch over the site of injury (Patch,n = 6); and 4) Cell group,hiPSCs-cardiomyocytes,-endothelial cells,and -smooth muscle cells (2 million,each) were injected into the injured myocardium pass through a fibrin patch (Cell+Patch,n = 5). At 4 weeks,the creatine phosphate (PCr)/ATP ratio,CK forward flux rate (Flux PCr→ATP),and k constant of CK forward flux rate (kPCr→ATP) were severely decreased at border zone myocardium (BZ) adjacent to MI. Cell treatment results in significantly increase of PCr/ATP ratio and improve the value of kPCr→ATP and Flux PCr→ATP in BZ myocardium. Moreover,the BZ myocardial CK total activity and protein expression of CK mitochondria isozyme and CK myocardial isozyme were significantly reduced,but recovered in response to cell treatment. Thus,cell therapy results in improvement of BZ bioenergetic abnormality in hearts with postinfarction LV remodeling,which is accompanied by significantly improvements in BZ CK activity and CK isozyme expression. The fast 2D 31P MR CSI mapping can reliably measure the heterogeneity of bioenergetics in hearts with post infarction LV remodeling. View Publication

The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine,pharmacological drug screening,and toxicity testing. However,full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study,we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 10(6) hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A,Wnt3a,and sodium butyrate to the culture medium. For further maturation,hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP),a marker for DE,was significantly (p textless 0.05) higher in 2D cultures,while secretion of albumin,a typical characteristic for mature hepatocytes,was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2,CYP2B6,and CYP3A4 in both groups,although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p textless 0.05) higher in 3D bioreactors compared with 2D cultures,which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin,cytokeratin 18 (CK18),and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition,cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures. View Publication

Human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs),dental pulp stem cells (hDPSCs) and bone marrow MSCs (hBMSCs) are exciting cell sources in regenerative medicine. However,there has been no report comparing hDPSCs,hBMSCs and hiPSC-MSCs for bone engineering in an injectable calcium phosphate cement (CPC) scaffold. The objectives of this study were to: (1) develop a novel injectable CPC containing hydrogel fibers encapsulating stem cells for bone engineering,and (2) compare cell viability,proliferation and osteogenic differentiation of hDPSCs,hiPSC-MSCs from bone marrow (BM-hiPSC-MSCs) and from foreskin (FS-hiPSC-MSCs),and hBMSCs in CPC for the first time. The results showed that the injection did not harm cell viability. The porosity of injectable CPC was 62%. All four types of cells proliferated and differentiated down the osteogenic lineage inside hydrogel fibers in CPC. hDPSCs,BM-hiPSC-MSCs,and hBMSCs exhibited high alkaline phosphatase,runt-related transcription factor,collagen I,and osteocalcin gene expressions. Cell-synthesized minerals increased with time (ptextless0.05),with no significant difference among hDPSCs,BM-hiPSC-MSCs and hBMSCs (ptextgreater0.1). Mineralization by hDPSCs,BM-hiPSC-MSCs,and hBMSCs inside CPC at 14d was 14-fold that at 1d. FS-hiPSC-MSCs were inferior in osteogenic differentiation compared to the other cells. In conclusion,hDPSCs,BM-hiPSC-MSCs and hBMSCs are similarly and highly promising for bone tissue engineering; however,FS-hiPSC-MSCs were relatively inferior in osteogenesis. The novel injectable CPC with cell-encapsulating hydrogel fibers may enhance bone regeneration in dental,craniofacial and orthopedic applications. View Publication

Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene,which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs,the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming,modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds,sodium butyrate (NaB) and Parnate,led to an increase in FXN expression and correction of repressive marks at the FXN locus,which persisted for several passages. However,prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore,we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together,these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications,thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA. View Publication

Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields,cellular heterogeneity,and limited scalability. In the present study,we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes,secreted hepatic proteins such as Albumin,Alpha Fetoprotein,and Fibrinogen,metabolized ammonia,and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes,such as LDL storage and uptake,ICG uptake and release,and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure,providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. View Publication

BACKGROUND Recently,accumulating evidence has shown that exosomes,the naturally secreted nanocarriers of cells,can exert therapeutic effects in various disease models in the absence of parent cells. However,application of exosomes in bone defect repair and regeneration has been rarely reported,and little is known regarding their underlying mechanisms. METHODS Exosomes derived from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-Exos) were combined with tricalcium phosphate (β-TCP) to repair critical-sized calvarial bone defects,and the efficacy was assessed by histological examination. We evaluated the in vitro effects of hiPSC-MSC-Exos on the proliferation,migration,and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by cell-counting,scratch assays,and qRT-PCR,respectively. Gene expression profiling and bioinformatics analyses were also used to identify the underlying mechanisms in the repair. RESULTS We found that the exosome/β-TCP combination scaffolds could enhance osteogenesis as compared to pure β-TCP scaffolds. In vitro assays showed that the exosomes could release from β-TCP and could be internalized by hBMSCs. In addition,the internalization of exosomes into hBMSCs could profoundly enhance the proliferation,migration,and osteogenic differentiation of hBMSCs. Furthermore,gene expression profiling and bioinformatics analyses demonstrated that exosome/β-TCP combination scaffolds significantly altered the expression of a network of genes involved in the PI3K/Akt signaling pathway. Functional studies further confirmed that the PI3K/Akt signaling pathway was the critical mediator during the exosome-induced osteogenic responses of hBMSCs. CONCLUSIONS We propose that the exosomes can enhance the osteoinductivity of β-TCP through activating the PI3K/Akt signaling pathway of hBMSCs,which means that the exosome/β-TCP combination scaffolds possess better osteogenesis activity than pure β-TCP scaffolds. These results indicate that naturally secreted nanocarriers-exosomes can be used as a bioactive material to improve the bioactivity of the biomaterials,and that hiPS-MSC-Exos combined with β-TCP scaffolds can be potentially used for repairing bone defects. View Publication

We report a method of high-speed phase contrast and bright field microscopy which permits large cell culture vessels to be scanned at much higher speed (up to 30 times faster) than when conventional methods are used without compromising image quality. The object under investigation moves continuously and is captured using a flash illumination which creates an exposure time short enough to prevent motion blur. During the scan the object always stays in focus due to a novel hardware-autofocus system. View Publication

BACKGROUND & AIMS One major obstacle of hepatitis B virus (HBV) research is the lack of efficient cell culture system permissive for viral infection and replication. The aim of our study was to establish a robust HBV infection model by using hepatocyte-like cells (HLCs) derived from human pluripotent stem cells. METHODS HLCs were differentiated from human embryonic stem cells and induced pluripotent stem cells. Maturation of hepatocyte functions was determined. After HBV infection,total viral DNA,cccDNA,total viral RNA,pgRNA,HBeAg and HBsAg were measured. RESULTS More than 90% of the HLCs expressed strong signals of human hepatocyte markers,like albumin,as well as known host factors required for HBV infection,suggesting that these cells possessed key features of mature hepatocytes. Notably,HLCs expressed the viral receptor sodium-taurocholate cotransporting polypeptide more stably than primary human hepatocytes (PHHs). HLCs supported robust infection and some spreading of HBV. Finally,by using this model,we identified two host-targeting agents,genistin and PA452,as novel antivirals. CONCLUSIONS Stem cell-derived HLCs fully support HBV infection. This novel HLC HBV infection model offers a unique opportunity to advance our understanding of the molecular details of the HBV life cycle; to further characterize virus-host interactions and to define new targets for HBV curative treatment. LAY SUMMARY Our study used human pluripotent stem cells to develop hepatocyte-like cells (HLCs) capable of expressing hepatocyte markers and host factors important for HBV infection. These cells fully support HBV infection and virus-host interactions,allowing for the identification of two novel antiviral agents. Thus,stem cell-derived HLCs provide a highly physiologically relevant system to advance our understanding of viral life cycle and provide a new tool for antiviral drug screening and development. View Publication