Amita M et al. (MAR 2013)
Proceedings of the National Academy of Sciences of the United States of America 110 13 E1212--E1221
Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4
Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development,but the soundness of this model has been challenged by others,who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7+ within 48 h,with minimal expression of mesoderm markers,including T (Brachyury). Instead,they begin to express a series of trophoblast markers,including HLA-G,demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium,and,over time,produce extensive amounts of human chorionic gonadotropin,progesterone,placental growth factor,and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling,which at day 2 provide colonies that are entirely KRT7+ and in which the majority of cells are transiently CDX2+. Colonies grown on two chemically defined media,including the one in which BMP4 was reported to drive mesoderm formation,also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts.
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Smagghe BJ et al. (MAR 2013)
PLoS ONE 8 3 e58601
MUC1* Ligand, NM23-H1, Is a Novel Growth Factor That Maintains Human Stem Cells in a More Naïve State
We report that a single growth factor,NM23-H1,enables serial passaging of both human ES and iPS cells in the absence of feeder cells,their conditioned media or bFGF in a fully defined xeno-free media on a novel defined,xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1�
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Pol SU et al. (SEP 2013)
Experimental Neurology 247 694--702
Sox10-MCS5 enhancer dynamically tracks human oligodendrocyte progenitor fate
In this study,we sought to establish a novel method to prospectively and dynamically identify live human oligodendrocyte precursor cells (OPCs) and oligodendrocyte lineage cells from brain dissociates and pluripotent stem cell culture. We selected a highly conserved enhancer element of the Sox10 gene,known as MCS5,which directs reporter expression to oligodendrocyte lineage cells in mouse and zebrafish. We demonstrate that lentiviral Sox10-MCS5 induced expression of GFP at high levels in a subpopulation of human CD140a/PDGF??R-sorted OPCs as well as their immature oligodendrocyte progeny. Furthermore,we show that almost all Sox10-MCS5:GFPhigh cells expressed OPC antigen CD140a and human OPCs expressing SOX10,OLIG2,and PDGFRA mRNAs could be prospectively identified using GFP based fluorescence activated cells sorting alone. Additionally,we established a human induced pluripotent cell (iPSC) line transduced with the Sox10-MCS5:GFP reporter using a Rex-Neo cassette. Similar to human primary cells,GFP expression was restricted to embryoid bodies containing both oligodendrocyte progenitor and oligodendrocyte cells and co-localized with NG2 and O4-positive cells respectively. As such,we have developed a novel reporter system that can track oligodendrocyte commitment in human cells,establishing a valuable tool to improve our understanding and efficiency of human oligodendrocyte derivation. ?? 2013 Elsevier Inc.
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Lu B et al. (MAY 2013)
Nature Neuroscience 16 5 562--570
Identification of NUB1 as a suppressor of mutant Huntingtin toxicity via enhanced protein clearance
Huntington's disease is caused by expanded CAG repeats in HTT,conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model,and 6 exhibited activity consistent with the in vitro screening results. Among these,negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment,interferon-β lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus,we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease.
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Brown HF et al. (JUN 2013)
Journal of Virology 87 12 7127--39
Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line
Herpesvirus saimiri (HVS) infects a range of human cell types with high efficiency. Upon infection,the viral genome can persist as high-copy-number,circular,nonintegrated episomes that segregate to progeny cells upon division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression in vitro and in vivo. Moreover,the HVS episome is able to persist and provide prolonged transgene expression during in vitro differentiation of mouse and human hemopoietic progenitor cells. Together,these properties are advantageous for induced pluripotent stem cell (iPSC) technology,whereby stem cell-like cells are generated from adult somatic cells by exogenous expression of specific reprogramming factors. Here we assess the potential of HVS-based vectors for the generation of induced pluripotent cancer stem-like cells (iPCs). We demonstrate that HVS-based exogenous delivery of Oct4,Nanog,and Lin28 can reprogram the Ewing's sarcoma family tumor cell line A673 to produce stem cell-like colonies that can grow under feeder-free stem cell culture conditions. Further analysis of the HVS-derived putative iPCs showed some degree of reprogramming into a stem cell-like state. Specifically,the putative iPCs had a number of embryonic stem cell characteristics,staining positive for alkaline phosphatase and SSEA4,in addition to expressing elevated levels of pluripotent marker genes involved in proliferation and self-renewal. However,differentiation trials suggest that although the HVS-derived putative iPCs are capable of differentiation toward the ectodermal lineage,they do not exhibit pluripotency. Therefore,they are hereby termed induced multipotent cancer cells.
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Caron NJ et al. (OCT 2013)
Biotechnology and Bioengineering 110 10 2706--2716
A human embryonic stem cell line adapted for high throughput screening
Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However,optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs,including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20,they remain highly useful. In particular,CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus,108 CA1S cells can be generated within one week from 106 cells to seed 106 wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm,a finding that was confirmed with H1 hESCs,although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines,these cells should ease the development of optimized hESC growth and differentiation protocols. In particular,they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses. Biotechnol. Bioeng. 2013;110: 2706–2716. textcopyright 2013 Wiley Periodicals,Inc.
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Sharma A et al. (JUN 2013)
Journal of Biological Chemistry 288 25 18439--18447
The role of SIRT6 protein in aging and reprogramming of human induced pluripotent stem cells
Aging is known to be the single most important risk factor for multiple diseases. Sirtuin 6,or SIRT6,has recently been identified as a critical regulator of transcription,genome stability,telomere integrity,DNA repair,and metabolic homeostasis. A knockout mouse model of SIRT6 has displayed dramatic phenotypes of accelerated aging. In keeping with its role in aging,we demonstrated that human dermal fibroblasts (HDFs) from older human subjects were more resistant to reprogramming by classic Yamanaka factors than those from younger human subjects,but the addition of SIRT6 during reprogramming improved such efficiency in older HDFs substantially. Despite the importance of SIRT6,little is known about the molecular mechanism of its regulation. We show,for the first,time posttranscriptional regulation of SIRT6 by miR-766 and inverse correlation in the expression of this microRNA in HDFs from different age groups. Our results suggest that SIRT6 regulates miR-766 transcription via a feedback regulatory loop,which has implications for the modulation of SIRT6 expression in reprogramming of aging cells.
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Gifford CA et al. (MAY 2013)
Cell 153 5 1149--1163
Transcriptional and epigenetic dynamics during specification of human embryonic stem cells
Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end,we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing,chromatin immunoprecipitation sequencing,and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition,we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches,leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions. ?? 2013 Elsevier Inc.
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Sundberg M et al. (AUG 2013)
Stem Cells 31 8 1548--1562
Improved cell therapy protocols for Parkinson's disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons
The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results,NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2,LMX1A,TH,GIRK2,PITX3,EN1,NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue,with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation,the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.
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Haraguchi Y et al. (DEC 2015)
Journal of Tissue Engineering and Regenerative Medicine 9 12 1363--1375
Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.
In this study,a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension,only a few aggregated cells were observed. However,after 3 days,culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry,immunocytochemistry and quantitative RT-PCR,and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium,expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore,the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A,BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes,including HCN4,MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes,including pacemakers. Moreover,when cardiac cell sheets were fabricated using differentiated cardiomyocytes,they beat spontaneously and synchronously,indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.
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Bioluminescence Imaging of Human Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Myocardial Infarction
Myocardial infarction is a leading cause of mortality and morbidity worldwide,and current treatments fail to address the underlying scarring and cell loss,which is a major cause of heart failure after infarction. The novel strategy,therapeutic angiogenesis and/or vasculogenesis with endothelial progenitor cells transplantation holds great promise to increase blood flow in ischemic areas,thus rebuild the injured heart and reverse the heart failure. Given the potential of self-renewal and differentiation into virtually all cell types,human embryonic stem cells (hESCs) may provide an alternate source of therapeutic cells by allowing the derivation of large numbers of endothelial cells for therapeutic angiogenesis and/or vasculogenesis of ischemic heart diseases. Moreover,to fully understand the fate of implanted hESCs or hESC derivatives,investigators need to monitor the motility of cells in living animals over time. In this chapter,we describe the application of bioluminescence reporter gene imaging to track the transplanted hESC-derived endothelial cells for treatment of myocardial infarction. The technology of inducing endothelial cells from hESCs will also be discussed.
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Lu S-J et al. (JUL 2013)
Regenerative medicine 8 4 413--424
3D microcarrier system for efficient differentiation of human pluripotent stem cells into hematopoietic cells without feeders and serum [corrected].
BACKGROUND Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential,a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation,the medium for 3D microcarriers was directly switched to EB medium. RESULTS hESCs on 3D microcarriers maintained pluripotency and formed EBs,which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating,EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore,this 3D system can also be adapted to human induced pluripotent stem cells,which generate functional hemangioblasts with high efficiency. CONCLUSION This 3D serum- and stromal-free microcarrier system is important for future clinical applications,with the potential of developing to a GMP-compatible scalable system.
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