技术资料

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

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

BACKGROUND Prenatal alcohol exposure (PAE) in animal models results in excitatory-inhibitory (E/I) imbalance in neocortex due to alterations in the GABAergic interneuron (IN) differentiation and migration. Thus,E/I imbalance is a potential cause for intellectual disability in individuals with fetal alcohol spectrum disorder (FASD),but whether ethanol (EtOH) changes glutamatergic and GABAergic IN specification during human development remains unknown. Here,we created a human cellular model of PAE/FASD and tested the hypothesis that EtOH exposure during differentiation of human pluripotent stem cell-derived neurons (hPSNs) would cause the aberrant production of glutamatergic and GABAergic neurons,resulting in E/I imbalance. METHODS We applied 50 mM EtOH daily to differentiating hPSNs for 50 days to model chronic first-trimester exposure. We used quantitative polymerase chain reaction,immunocytochemical,and electrophysiological analysis to examine the effects of EtOH on hPSN specification and functional E/I balance. RESULTS We found that EtOH did not alter neural induction nor general forebrain patterning and had no effect on the expression of markers of excitatory cortical pyramidal neurons. In contrast,our data revealed highly significant changes to levels of transcripts involved with IN precursor development (e.g.,GSX2,DLX1/2/5/6,NR2F2) as well as mature IN specification (e.g.,SST,NPY). Interestingly,EtOH did not affect the number of GABAergic neurons generated nor the frequency or amplitude of miniature excitatory and inhibitory postsynaptic currents. CONCLUSIONS Similar to in vivo rodent studies,EtOH significantly and specifically altered the expression of genes involved with IN specification from hPSNs,but did not cause imbalances of synaptic excitation-inhibition. Thus,our findings corroborate previous studies pointing to aberrant neuronal differentiation as an underlying mechanism of intellectual disability in FASD. However,in contrast to rodent binge models,our chronic exposure model suggests possible compensatory mechanisms that may cause more subtle defects of network processing rather than gross alterations in total E/I balance. View Publication

Alternative models for more rapid compound safety testing are of increasing demand. With emerging techniques using human pluripotent stem cells,the possibility of generating human in vitro models has gained interest,as factors related to species differences could be potentially eliminated. When studying potential neurotoxic effects of a compound it is of crucial importance to have both neurons and glial cells. We have successfully developed a protocol for generating in vitro 3D human neural tissues,using neural progenitor cells derived from human embryonic stem cells. These 3D neural tissues can be maintained for two months and undergo progressive differentiation. We showed a gradual decreased expression of early neural lineage markers,paralleled by an increase in markers specific for mature neurons,astrocytes and oligodendrocytes. At the end of the two-month culture period the neural tissues not only displayed synapses and immature myelin sheaths around axons,but electrophysiological measurements also showed spontaneous activity. Neurotoxicity testing - comparing non-neurotoxic to known neurotoxic model compounds - showed an expected increase in the marker of astroglial reactivity after exposure to known neurotoxicants methylmercury and trimethyltin. Although further characterization and refinement of the model is required,these results indicate its potential usefulness for in vitro neurotoxicity testing. View Publication

A progressive increase in MECP2 protein levels is a crucial and precisely regulated event during neurodevelopment,but the underlying mechanism is unclear. We report that MECP2 is regulated post-transcriptionally during in vitro differentiation of human embryonic stem cells (hESCs) into cortical neurons. Using reporters to identify functional RNA sequences in the MECP2 3' UTR and genetic manipulations to explore the role of interacting factors on endogenous MECP2,we discover combinatorial mechanisms that regulate RNA stability and translation. The RNA-binding protein PUM1 and pluripotent-specific microRNAs destabilize the long MECP2 3' UTR in hESCs. Hence,the 3' UTR appears to lengthen during differentiation as the long isoform becomes stable in neurons. Meanwhile,translation of MECP2 is repressed by TIA1 in hESCs until HuC predominates in neurons,resulting in a switch to translational enhancement. Ultimately,3' UTR-directed translational fine-tuning differentially modulates MECP2 protein in the two cell types to levels appropriate for normal neurodevelopment. View Publication

Behçet's disease (BD) is a chronic inflammatory and multisystemic autoimmune disease of unknown etiology. Due to the lack of a specific test for BD,its diagnosis is very difficult and therapeutic options are limited. Induced pluripotent stem cell (iPSC) technology,which provides inaccessible disease-relevant cell types,opens a new era for disease treatment. In this study,we generated BD iPSCs from patient somatic cells and differentiated them into hematopoietic precursor cells (BD iPSC-HPCs) as BD model cells. Based on comparative transcriptome analysis using our BD model cells,we identified eight novel BD-specific genes,AGTR2,CA9,CD44,CXCL1,HTN3,IL-2,PTGER4,and TSLP,which were differentially expressed in BD patients compared with healthy controls or patients with other immune diseases. The use of CXCL1 as a BD biomarker was further validated at the protein level using both a BD iPSC-HPC-based assay system and BD patient serum samples. Furthermore,we show that our BD iPSC-HPC-based drug screening system is highly effective for testing CXCL1 BD biomarkers,as determined by monitoring the efficacy of existing anti-inflammatory drugs. Our results shed new light on the usefulness of patient-specific iPSC technology in the development of a benchmarking platform for disease-specific biomarkers,phenotype- or target-driven drug discovery,and patient-tailored therapies. 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

In the past decade,tissue microarray (TMA) technology has evolved as an innovative tool for high-throughput proteomics analysis and mainly for biomarker validation. Similarly,enormous amount of data can be obtained from the cell line macroarray (CLMA) technology,which developed from the TMA using formalin-fixed,paraffin-embedded cell pellets. Here,we applied CLMA technology in stem cell research and in particular to identify bona fide neogenerated human induced pluripotent stem cell (hiPSC) clones suitable for down the line differentiation. All hiPSC protocols generate tens of clones,which need to be tested to determine genetically stable cell lines suitable for differentiation. Screening methods generally rely on fluorescence-activated cell sorting isolation and coverslip cell growth followed by immunofluorescence; these techniques could be cumbersome. Here,we show the application of CLMA to identify neogenerated pluripotent cell colonies and neuronal differentiated cell products. We also propose the use of the automated image analyzer,TissueQuest,as a reliable tool to quickly select the best clones,based upon the level of expression of multiple pluripotent biomarkers. View Publication