技术资料

Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However,marker-free genome editing using standard protocols remains inefficient,yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing,so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA),donor DNA and piggyBac transposase resulted in efficient,targeted genome editing and concurrent scarless transgene excision. Using this approach,in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%. View Publication

Human embryonic stem cell (hESC) line chHES-480 was derived from abnormal blastocyst diagnosed with adrenoleukodystrophy (ALD) after preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that chHES-480 cell line carried a hemizygous missense mutation c.1825GtextgreaterA(p.Glu609Lys) of ABCD1 gene. Characteristic tests proved that the chHES-480 cell line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. View Publication

The gene of ATP-binding cassette subfamily C member 8 (Abcc8) is cytogenetically located at 11p15.1 and encodes the sulfonylurea receptor (SUR1). SUR1 is a subunit of ATP-sensitive potassium channel (KAPT) in the β-cell regulating insulin secretion. Mutations of ABCC8 are responsible for congenital hyperinsulinism (CHI). Here we reported that an Abcc8 heterozygous mutant cell line was generated by CRISPR/Cas9 technique with 1bp insertion resulting in abnormal splicing on human embryonic stem cell line H1. The phenotypic characteristics of this cell line reveal defective KATP channel and diazoxide-responsive that provides ideal model for molecular pathology research and drug screening for CHI. View Publication

Induced pluripotent stem cells (iPSCs) are being pursued as a source of cells for autologous therapies,many of which will be aimed at aged patients. To explore the impact of age on iPSC quality,we produced iPSCs from blood cells of 16 donors aged 21-100. We find that iPSCs from older donors retain an epigenetic signature of age,which can be reduced through passaging. Clonal expansion via reprogramming also enables the discovery of somatic mutations present in individual donor cells,which are missed by bulk sequencing methods. We show that exomic mutations in iPSCs increase linearly with age,and all iPSC lines analyzed carry at least one gene-disrupting mutation,several of which have been associated with cancer or dysfunction. Unexpectedly,elderly donors (textgreater90 yrs) harbor fewer mutations than predicted,likely due to a contracted blood progenitor pool. These studies establish that donor age is associated with an increased risk of abnormalities in iPSCs and will inform clinical development of reprogramming technology. View Publication

Success in the differentiating human embryonic stem cells (hESCs) into insulin-secreting β cells raises new hopes for diabetes treatment. In this work,we demonstrated the feasibility of developing islet organoids from hESCs within biomimetic 3D scaffolds. We showed that such a 3D microenvironment is critical to the generation of pancreatic endoderm and endocrine from hESCs. The organoids formed consisted of pancreatic α,β,δ,and pancreatic polypeptide (PP) cells. A high-level co-expression of PDX1,NKX6.1,and NGN3 in these cells suggests the characteristics of pancreatic β cells. More importantly,most insulin-secreting cells generated did not express glucagon,somatostatin,or PP. The expression of mature β cell marker genes such as Pdx1,Ngn3,Insulin,MafA,and Glut2 was detected in these 3D-induced cell clusters. A high-level expression of C-peptide confirmed the de novo endogenous insulin production in these 3D induced cells. Insulin-secretory granules,an indication of β cell maturity,were detected in these cells as well. Glucose challenging experiments suggested that these cells are sensitive to glucose levels due to their elevated maturity. Exposing the cells to a high concentration of glucose induced a sharp increase in insulin secretion. View Publication

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However,the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p,miR-199a-3p,and miR-214 in the microRNA gene cluster. Meanwhile,the expression levels of their targets related to regulation of hypoxia-stimulated cell migration,such as HIF1A,MET,and MAPK1,were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy. View Publication

Importance Human motor neurons may be reliably derived from induced pluripotent stem cells (iPSCs). In vivo transplant studies of human iPSCs and their cellular derivatives are essential to gauging their clinical utility. Objective To determine whether human iPSC-derived motor neurons can engraft in an immunodeficient mouse model of sciatic nerve injury. Design,Setting,and Subjects This nonblinded interventional study with negative controls was performed at a biomedical research institute using an immunodeficient,transgenic mouse model. Induced pluripotent stem cell-derived motor neurons were cultured and differentiated. Cells were transplanted into 32 immunodeficient mice with sciatic nerve injury aged 6 to 15 weeks. Tissue analysis was performed at predetermined points after the mice were killed humanely. Animal experiments were performed from February 24,2015,to May 2,2016,and data were analyzed from April 7,2015,to May 27,2016. Interventions Human iPSCs were used to derive motor neurons in vitro before transplant. Main Outcomes and Measures Evidence of engraftment based on immunohistochemical analysis (primary outcome measure); evidence of neurite outgrowth and neuromuscular junction formation (secondary outcome measure); therapeutic effect based on wet muscle mass preservation and/or electrophysiological evidence of nerve and muscle function (exploratory end point). Results In 13 of the 32 mice undergoing the experiment,human iPSC-derived motor neurons successfully engrafted and extended neurites to target denervated muscle. Human iPSC-derived motor neurons reduced denervation-induced muscular atrophy (mean [SD] muscle mass preservation,54.2% [4.0%]) compared with negative controls (mean [SD] muscle mass preservation,33.4% [2.3%]) (P = .04). No electrophysiological evidence of muscle recovery was found. Conclusions and Relevance Human iPSC-derived motor neurons may have future use in the treatment of peripheral motor nerve injury,including facial paralysis. Level of Evidence NA. View Publication

BACKGROUND Oesophageal squamous cell carcinoma (ESCC) and colorectal cancer (CRC) are common types of human cancer. Spheroid colony formation is used to characterize cancer stem cell (CSCs). In the present study,we analyzed the significance of kinesin family 11 (KIF11 in human ESCC and CRC. MATERIALS AND METHODS Expression of KIF11 in 105 ESCC and 100 CRC cases was determined using immunohistochemistry. RNA interference was used to inhibit KIF11 expression in ESCC and CRC cell lines. RESULTS In total,61 out of 105 (58%) ESCC and 62 out of 100 (62%) CRC cases were positive for KIF11. Expression of KIF11 was not associated with any clinicopathological characteristics. Both the number and size of spheres produced by from TE-5 ESCC cells and DLD-1 CRC cells were significantly reduced upon KIF11 siRNA transfection compared to negative control siRNA transfection. CONCLUSION These results indicate that KIF11 plays an important role in CSCs of ESCC and CRC. View Publication

Can photothermal gold nanoparticle mediated laser manipulation be applied to induce cardiac contraction? Based on our previous work,we present a novel concept of cell stimulation. A 532 nm picosecond laser was employed to heat gold nanoparticles on cardiomyocytes. This leads to calcium oscillations in the HL-1 cardiomyocyte cell line. As calcium is connected to the contractility,we aimed to alter the contraction rate of native and stem cell derived cardiomyocytes. A contraction rate increase was particularly observed in calcium containing buffer with neonatal rat cardiomyocytes. Consequently,the study provides conceptual ideas for a light based,nanoparticle mediated stimulation system. View Publication

Engineered neural stem cells (NSCs) are a promising approach to treating glioblastoma (GBM). The ideal NSC drug carrier for clinical use should be easily isolated and autologous to avoid immune rejection. We transdifferentiated (TD) human fibroblasts into tumor-homing early-stage induced NSCs (h-iNSC(TE)),engineered them to express optical reporters and different therapeutic gene products,and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSC(TE) in patient-derived GBM models of surgical and nonsurgical disease. Molecular and functional analysis revealed that our single-factor SOX2 TD strategy converted human skin fibroblasts into h-iNSC(TE) that were nestin(+) and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSC(TE) rapidly migrated to human GBM cells and penetrated human GBM spheroids,a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSC(TE) delivery of the proapoptotic agent tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) reduced the size of solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. Additionally,h-iNSC(TE) thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSC(TE)-TK) reduced the size of patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical NSC therapy,h-iNSC(TE)-TK therapy delivered into the postoperative surgical resection cavity delayed the regrowth of residual GBMs threefold and prolonged survival from 46 to 60 days. These results suggest that TD of human skin into h-iNSC(TE) is a platform for creating tumor-homing cytotoxic cell therapies for cancer,where the potential to avoid carrier rejection could maximize treatment durability in human trials. View Publication

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-,atrial-,and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives,which in turn promoted cardiomyocyte differentiation. Moreover,a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation,improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation,measuring action potentials,and intracellular Ca(2+) dynamics. These findings are relevant for improving our understanding on human heart development,and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes,similar to those observed in adult cardiac myocytes. View Publication

Although human induced pluripotent stem cells (hiPSCs) hold great potential for the study of human diseases affecting disparate cell types,they have been underutilized in seeking mechanistic insights into the pathogenesis of congenital craniofacial disorders. Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in EFNB1 and characterized by craniofacial,skeletal,and neurological anomalies. Heterozygous females are more severely affected than hemizygous males,a phenomenon termed cellular interference that involves mosaicism for EPHRIN-B1 function. Although the mechanistic basis for cellular interference in CFNS has been hypothesized to involve Eph/ephrin-mediated cell segregation,no direct evidence for this has been demonstrated. Here,by generating hiPSCs from CFNS patients,we demonstrate that mosaicism for EPHRIN-B1 expression induced by random X inactivation in heterozygous females results in robust cell segregation in human neuroepithelial cells,thus supplying experimental evidence that Eph/ephrin-mediated cell segregation is relevant to pathogenesis in human CFNS patients. View Publication