技术资料

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript,we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore,we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages,further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication

The integration of up- and downstream unit operations can result in the elimination of hold steps,thus decreasing the footprint,and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC),where high numbers of pure cells,at low volumes,need to be delivered for therapy applications. This study reports a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. Moreover,we have also explored a continuous alternative for concentrating hMSC. Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio,and led to a shift in cells' metabolism. TFF operated either in continuous or discontinuous allowed to concentrate cells,with high cell recovery (>80%) and viability (>95%); furthermore,continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration led to higher protein clearance (98%) with lower cell death,when comparing to discontinuous diafiltration. Overall,an integrated process allowed for a shorter process time,recovering 70% of viable hMSC (>95%),with no changes in terms of morphology,immunophenotype,proliferation capacity and multipotent differentiation potential. View Publication

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However,current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation,which is required to generate endodermal,ectodermal,and mesodermal cell lineages. We report a new,straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs,which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1,GAG,and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats,magnetic resonance imaging studies showed gradual engraftment,and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs,which could improve cartilage regeneration outcomes in arthritic joints. View Publication

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However,tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs,especially those in cancer patients. To circumvent these issues,we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable,but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition,invasion,stemness,and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ,downstream protumor factors,and hyaluronan and its cofactor TSG6,which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for off-the-shelf" therapies and bioengineering applications." View Publication

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton,cornea,peripheral nervous system,and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs),such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),further modifications are required to improve the robustness,efficacy,and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions,the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70-80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons,glia,melanocytes,and corneal endothelial cells. In addition,cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine. View Publication

Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis,and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein,we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly,TRAIL treatment induced RANKL mRNA expression in these cells. In addition,TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition,siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus,our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells. View Publication

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids,rather than two-dimensional (2D) monolayers,is advantageous for many regenerative,biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly,we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells,where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. View Publication

Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically,various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods,we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods,which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming,thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells. View Publication

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report,human mesenchymal cells (hMSCs) generated from the human ES cell line H9,were reprogrammed back to induced pluripotent state using Oct-4,Sox2,Nanog,and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes,lipid droplets,glycogen,scarce reticulum) and nuclear levels (features of nuclear plasticity,presence of euchromatin,reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal-epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. View Publication

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with $$-thalassemia ($$-thal) with the aim to generate trangene-free $$-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4,Klf4,Sox2,cMyc,and Lin28 resulted in formation of five iPSC colonies,from which three were picked up and expanded in $$-thal-iPSC lines. After 10 serial passages in vitro,$$-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs,whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%,but with a decreased hematopoietic colony-forming capability. In conclusion,we report herein the generation of transgene-free $$-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover,it was demonstrated that the mRNA-based reprogramming method,used mainly in fibroblasts,is also suitable for reprogramming of human BM-MSCs. View Publication

BACKGROUND Cardiovascular cell therapy represents a promising field,with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study (Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease") consists of fresh mononuclear cells (MNC) isolated from autologous bone marrow (BM) through density gradient centrifugation on standard Ficoll-Paque. Cells are tested for safety (sterility� View Publication

AIM To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics,colony-forming potential,as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS hESC-MSC showed similar morphology,and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications. View Publication