技术资料

Despite significant advances in commercially available media and kits and the differentiation approaches for human neural stem cell (NSC) generation,NSC production from the differentiation of human pluripotent stem cell (hPSC) is complicated by its time-consuming procedure,complex medium composition,and purification step. In this study,we developed a convenient and simplified NSC production protocol to meet the demand of NSC production. We demonstrated that NSCs can be generated efficiently without requirement of specific small molecules or embryoid body formation stage. Our experimental results suggest that a short suspension culture period may facilitate ectoderm lineage specification rather than endoderm or mesoderm lineage specification from hPSCs. The method developed in this study shortens the turnaround time of NSC production from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) differentiation. It provides a straightforward and useful strategy for generating NSCs that can benefit a wide range of research applications for human brain research. View Publication

Improvement of methods to produce endoderm-derived cells from pluripotent stem cells is important to realize high-efficient induction of endodermal tissues such as pancreas and hepatocyte. Difficulties hampering such efforts include the low efficiency of definitive endoderm cell induction and establishing appropriate defined culture conditions to ensure a safe cell source for human transplantation. Based on previous studies,we revised the experimental condition of definitive endoderm induction in feeder- and serum-free culture. Our results suggested that CHIR99021 is more effective than Wnt3A ligand in feeder- and serum-free conditions. In addition,keeping cell density low during endoderm induction is important for the efficiency. On the other hand,we showed that overtreatment with CHIR99021 converted the cells into BRACHYURY-expressing posterior mesoderm cells rather than endoderm,indicating strict CHIR99021 treatment requirements for endoderm differentiation. Nevertheless,these results should enable better control in the production of definitive endoderm-derived cells. View Publication

The development of novel platforms for large scale production of human embryonic stem cells (hESC) derived cardiomyocytes (CM) becomes more crucial as the demand for CMs in preclinical trials,high throughput cardio toxicity assays and future regenerative therapeutics rises. To this end,we have designed a microcarrier (MC) suspension agitated platform that integrates pluripotent hESC expansion followed by CM differentiation in a continuous,homogenous process.Hydrodynamic shear stresses applied during the hESC expansion and CM differentiation steps drastically reduced the capability of the cells to differentiate into CMs. Applying vigorous stirring during pluripotent hESC expansion on Cytodex 1 MC in spinner cultures resulted in low CM yields in the following differentiation step (cardiac troponin-T (cTnT): 22.83. ??. 2.56%; myosin heavy chain (MHC): 19.30. ??. 5.31%). Whereas the lower shear experienced in side to side rocker (wave type) platform resulted in higher CM yields (cTNT: 47.50. ??. 7.35%; MHC: 42.85. ??. 2.64%). The efficiency of CM differentiation is also affected by the hydrodynamic shear stress applied during the first 3. days of the differentiation stage. Even low shear applied continuously by side to side rocker agitation resulted in very low CM differentiation efficiency (cTnT. textless. 5%; MHC. textless. 2%). Simply by applying intermittent agitation during these 3. days followed by continuous agitation for the subsequent 9. days,CM differentiation efficiency can be substantially increased (cTNT: 65.73. ??. 10.73%; MHC: 59.73. ??. 9.17%). These yields are 38.3% and 39.3% higher (for cTnT and MHC respectively) than static culture control.During the hESC expansion phase,cells grew on continuously agitated rocker platform as pluripotent cell/MC aggregates (166??88??105??m2) achieving a cell concentration of 3.74??0.55??106cells/mL (18.89??2.82 fold expansion) in 7days. These aggregates were further differentiated into CMs using a WNT modulation differentiation protocol for the subsequent 12days on a rocking platform with an intermittent agitation regime during the first 3days. Collectively,the integrated MC rocker platform produced 190.5??58.8??106 CMs per run (31.75??9.74 CM/hESC seeded). The robustness of the system was demonstrated by using 2 cells lines,hESC (HES-3) and human induced pluripotent stem cell (hiPSC) IMR-90. The CM/MC aggregates formed extensive sarcomeres that exhibited cross-striations confirming cardiac ontogeny. Functionality of the CMs was demonstrated by monitoring the effect of inotropic drug,Isoproterenol on beating frequency.In conclusion,we have developed a simple robust and scalable platform that integrates both hESC expansion and CM differentiation in one unit process which is capable of meeting the need for large amounts of CMs. ?? 2014. View Publication

Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet,cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation,viability and differentiation potential. Here,a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation,mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover,the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics. View Publication

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells,retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE),and fetal RPE. The protocols include collection of RNA for analysis by microarray,and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally,cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types. View Publication

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a disease in a dish" model of human neuromuscular diseases. Major hurdles View Publication

Background: Human induced pluripotent stem cells,which can be differentiated into hepatocyte-like cells,could provide a source for liver regeneration and bio-artificial liver devices. However,the functionality of hepatocyte-like cells is significantly lower than that of primary hepatocytes. Aims: To investigate whether serum from patients undergoing hepatectomy might promote differentiation from human induced pluripotent stem cells to hepatocyte-like cells. Methods: Serum from patients undergoing hepatectomy (acquired pre-hepatectomy and 3. hours,1 day and 3 days post-hepatectomy) was used to replace foetal bovine serum when differentiating human induced pluripotent stem cells into hepatocyte-like cells. Properties of hepatocyte-like cells were assessed and compared with cells cultured in foetal bovine serum. Results: The differentiation efficiency and functionality of hepatocyte-like cells cultured in human serum 3. hours and 1 day post-hepatectomy were superior to those cultured in foetal bovine serum and human serum pre-hepatectomy. Human serum 3 days post-hepatectomy had an equal effect to that of human serum pre-hepatectomy. Some cytochrome P450 isozyme transcript levels of hepatocyte-like cells cultured in human serum were higher than those cultured in foetal bovine serum. Conclusion: Human serum,particularly that acquired relatively soon after hepatectomy,can enhance the differentiation efficiency and functionality of hepatocyte-like cells derived from human induced pluripotent stem cells. textcopyright 2014 Editrice Gastroenterologica Italiana S.r.l. View Publication

The pluripotent property of hESCs makes them attractive for treatment of degenerative diseases such as diabetes. We have developed a stage-wise directed differentiation protocol to produce alginate-encapsulated islet-like cells derived from hESCs,which can be directly implanted for diabetes therapy. The advantage of alginate encapsulation lies in its capability to immunoisolate,along with the added possibility of scalable culture. We have evaluated the possibility of encapsulating hESCs at different stages of differentiation. Encapsulation of predifferentiated cells resulted in insufficient cellular yield and differentiation. On the other hand,encapsulation of undifferentiated hESCs followed by differentiation induction upon encapsulation,resulted in the highest viability and differentiation. More striking was that alginate encapsulation resulted in a much stronger differentiation compared to parallel 2D cultures,resulting in 20-fold increase in c-peptide protein synthesis. To elucidate the mechanism contributing to encapsulation-mediated enhancement in hESC maturation,investigation of the signaling pathways revealed interesting insight. While the phospho-protein levels of all the tested signaling molecules were lower under encapsulation,the ratio of pSMAD/pAKT was significantly higher,indicating a more efficient signal transduction under encapsulation. These results clearly demonstrate that alginate encapsulation of hESCs and differentiation to islet-cells types provides a potentially translatable treatment option for type1 diabetes. View Publication

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless,clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally,derivation of hiPSCs should be rapid and efficient in order to minimize manipulations,reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here,we provide an optimized,fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity,purity,stability and safety at a GMP facility and cryopreserved. To our knowledge,as a proof of principle,these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes. View Publication

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased,genetic,and phenotypic representations. In this study,we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a do-it-yourself" basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming View Publication

One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation,whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans,or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs,suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci,characteristic of the inactive X. Moreover,analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs. View Publication

Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development,modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (˜70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (textgreater250%) MN production in chemically defined adherent cultures. Furthermore,we show that Islet-1 is essential for formation of mature and functional human MNs,but,unlike its mouse counterpart,does not regulate cell survival or suppress the V2a interneuron fate. Together,our discoveries improve the strategy for MN derivation,advance our understanding of human neural specification and MN development,and provide invaluable tools for human developmental studies,drug discovery and regenerative medicine. View Publication