Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection

The protocols described here address methods used in two crucial stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. The first is an optimized method for producing lentivirus at an efficiency 600-fold greater than previously published, and it includes conjugation of the lentivirus to streptavidin superparamagnetic particles; this process takes 8 d. The second method enables the isolation of true hiPSCs immediately after somatic cell reprogramming and involves column-based positive selection of cells expressing the pluripotency marker TRA-1-81. This process takes 2 h and, as it is directly compatible with feeder-free culture, the time burden of manually identifying and mechanically propagating hiPSC colonies is reduced drastically. Taken together, these methods accelerate the production of hiPSCs and enable lines to be isolated, expanded to approxiamtely 10? cells and cryopreserved within 6-8 weeks.     

A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation

Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines.     

Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.     

基于人多潜能干细胞的人类疾病研究与治疗

人多潜能干细胞(hPSC)包括人胚胎干细胞(hESC)和诱导性多潜能干细胞(hiPSC),理论上具有分化成为人类所有细胞类型的能力.基于hPSC的基因打靶技术,不但可以纠正人基因组中的遗传突变用于细胞治疗,还可以通过反向遗传学的方式向hPSC引入疾病特异的突变.将携带人类疾病遗传基因的hPSC分化为特定的细胞类型,在理论上可以在体外模拟人类疾病的发生,研究人类疾病发生的机理,并建立体外筛选平台寻找治疗性药物.基因编辑和干细胞技术的结合将为人类疾病的机制研究和再生医学治疗带来革命性的突破.     

综述:基于人多潜能干细胞的人类疾病研究与治疗

人多潜能干细胞(hPSC)包括人胚胎干细胞(hESC)和诱导性多潜能干细胞(hiPSC),理论上具有分化成为人类所有细胞类型的能力.基于hPSC的基因打靶技术,不但可以纠正人基因组中的遗传突变用于细胞治疗,还可以通过反向遗传学的方式向hPSC引入疾病特异的突变.将携带人类疾病遗传基因的hPSC分化为特定的细胞类型,在理论上可以在体外模拟人类疾病的发生,研究人类疾病发生的机理,并建立体外筛选平台寻找治疗性药物.基因编辑和干细胞技术的结合将为人类疾病的机制研究和再生医学治疗带来革命性的突破.     

Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors

Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ～4 weeks.     

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate ^1-3. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich''s ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged ^4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich''s ataxia patients and control individuals ⁶, human newborn fibroblasts, as well as human keratinocytes.     

miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells

The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EPI-SCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR-371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior.     

Sialyl-lactotetra,a Novel Cell Surface Marker of Undifferentiated Human Pluripotent Stem Cells

Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10⁹ cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells.     

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.     

Somatic cell reprogramming for regenerative medicine: SCNT vs. iPS cells

Reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. However, a debate over which method is better, somatic cell nuclear transfer (SCNT) or induced pluripotent stem (iPS) cells, still persists. Both approaches have the potential to generate patient-specific pluripotent stem cells for replacement therapy. Yet, although SCNT has been successfully applied in various vertebrates, no human pluripotent stem cells have been generated by SCNT due to technical, legal and ethical difficulties. On the other hand, human iPS cell lines have been reported from both healthy and diseased individuals. A recent study reported the generation of triploid human pluripotent stem cells by transferring somatic nuclei into oocytes, a variant form of SCNT. In this essay, we discuss this progress and the potentials of these two reprogramming approaches for regenerative medicine.     

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.     

Copy number variations (CNVs) in human pluripotent cell-derived neuroprogenitors

Results from the analysis of copy number variations (CNVs) in human pluripotent cell-derived neuroprogenitor cell lines (hiPSC and hESC-derived NPC) are presented. Two different types of CNVs were detected: a) CNVs inherited from the original source of pluripotent cells (hESC and hiPSC) and b) CNVs detected either in the original source of pluripotent cells or in the derived NPC cell lines but not in both at the same time. Our data suggest that submicroscopic chromosomal changes happened during culture and manipulation of cells and those differentiation procedures could result in gains and losses of genomic regions in pluripotent cell-derived neuroprogenitors. Overall, the results indicate that even chromosomally stable stem cell lines would need to be analyzed in detail by high resolution methodologies before their clinical use.     

Gi-Coupled GPCR Signaling Controls the Formation and Organization of Human Pluripotent Colonies

Background

Reprogramming adult human somatic cells to create human induced pluripotent stem (hiPS) cell colonies involves a dramatic morphological and organizational transition. These colonies are morphologically indistinguishable from those of pluripotent human embryonic stem (hES) cells. G protein-coupled receptors (GPCRs) are required in diverse developmental processes, but their role in pluripotent colony morphology and organization is unknown. We tested the hypothesis that G_i-coupled GPCR signaling contributes to the characteristic morphology and organization of human pluripotent colonies.

Methodology/Principal Findings

Specific and irreversible inhibition of G_i-coupled GPCR signaling by pertussis toxin markedly altered pluripotent colony morphology. Wild-type hES and hiPS cells formed monolayer colonies, but colonies treated with pertussis toxin retracted inward, adopting a dense, multi-layered conformation. The treated colonies were unable to reform after a scratch wound insult, whereas control colonies healed completely within 48 h. In contrast, activation of an alternative GPCR pathway, G_s-coupled signaling, with cholera toxin did not affect colony morphology or the healing response. Pertussis toxin did not alter the proliferation, apoptosis or pluripotency of pluripotent stem cells.

Conclusions/Significance

Experiments with pertussis toxin suggest that G_i signaling plays a critical role in the morphology and organization of pluripotent colonies. These results may be explained by a G_i-mediated density-sensing mechanism that propels the cells radially outward. GPCRs are a promising target for modulating the formation and organization of hiPS and hES cell colonies and may be important for understanding somatic cell reprogramming and for engineering pluripotent stem cells for therapeutic applications.     

基因修饰人多能干细胞的高效单克隆建系方法

目标:提供一种能够显著提高慢病毒稳定转染人多能干细胞的方法,并建立一种简便无损的转染细胞筛选方法.方法:在慢病毒转染人多能干细胞过程,分别比较添加与不添加Y-27632情况下细胞形态的动态变化规律,以及细胞不同形态下对慢病毒颗粒的摄入能力差异,优化建立高效的慢病毒转染方法.随后,设计并研制可视化的简便显微操作装置,探索...