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  • Enhancing Hematopoiesis from Murine Embryonic Stem Cells through MLL1-Induced Activation of a Rac/Rho/Integrin Signaling Axis
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-16
    Weiwei Yang; G. Devon Trahan; Elizabeth D. Howell; Nancy A. Speck; Kenneth L. Jones; Austin E. Gillen; Kent Riemondy; Jay Hesselberth; David Bryder; Patricia Ernst
  • Force and Calcium Transients Analysis in Human Engineered Heart Tissues Reveals Positive Force-Frequency Relation at Physiological Frequency
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-16
    Umber Saleem; Ingra Mannhardt; Ingke Braren; Chris Denning; Thomas Eschenhagen; Arne Hansen

    Force measurements in ex vivo and engineered heart tissues are well established. Analysis of calcium transients (CaT) is complementary to force, and the combined analysis is meaningful to the study of cardiomyocyte biology and disease. This article describes a model of human induced pluripotent stem cell cardiomyocyte-derived engineered heart tissues (hiPSC-CM EHTs) transduced with the calcium sensor GCaMP6f followed by sequential analysis of force and CaT. Average peak analysis demonstrated the temporal sequence of the CaT preceding the contraction twitch. The pharmacological relevance of the test system was demonstrated with inotropic indicator compounds. Force-frequency relationship was analyzed in the presence of ivabradine (300 nM), which reduced spontaneous frequency and unmasked a positive correlation of force and CaT at physiological human heart beating frequency with stimulation frequency between 0.75 and 2.5 Hz (force +96%; CaT +102%). This work demonstrates the usefulness of combined force/CaT analysis and demonstrates a positive force-frequency relationship in hiPSC-CM EHTs.

  • Toward Guidelines for Research on Human Embryo Models Formed from Stem Cells
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-16
    Insoo Hyun; Megan Munsie; Martin F. Pera; Nicolas C. Rivron; Janet Rossant

    Over the past few years, a number of research groups have reported striking progress on the generation of in vitro models from mouse and human stem cells that replicate aspects of early embryonic development. Not only do these models reproduce some key cell fate decisions but, especially in the mouse system, they also mimic the spatiotemporal arrangements of embryonic and extraembryonic tissues that are required for developmental patterning and implantation in the uterus. If such models could be developed for the early human embryo, they would have great potential benefits for understanding early human development, for biomedical science, and for reducing the use of animals and human embryos in research. However, guidelines for the ethical conduct of this line of work are at present not well defined. In this Forum article, we discuss some key aspects of this emerging area of research and provide some recommendations for its ethical oversight.

  • iPSC-Based Modeling of RAG2 Severe Combined Immunodeficiency Reveals Multiple T Cell Developmental Arrests
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-16
    Maria Themeli; Amiet Chhatta; Hester Boersma; Henk Jan Prins; Martijn Cordes; Edwin de Wilt; Aïda Shahrabi Farahani; Bart Vandekerckhove; Mirjam van der Burg; Rob C. Hoeben; Frank J.T. Staal; Harald M.M. Mikkers
  • Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-09
    Hui-Hsuan Kuo; Xiaozhi Gao; Jean-Marc DeKeyser; K. Ashley Fetterman; Emily A. Pinheiro; Carly J. Weddle; Hananeh Fonoudi; Michael V. Orman; Marisol Romero-Tejeda; Mariam Jouni; Malorie Blancard; Tarek Magdy; Conrad L. Epting; Alfred L. George; Paul W. Burridge

    Human induced pluripotent stem cell (hiPSC) culture has become routine, yet the cost of pluripotent cell media, frequent medium changes, and the reproducibility of differentiation have remained restrictive. Here, we describe the formulation of a hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. The reagents in B8 represent only 3% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: fibroblast growth factor 2, transforming growth factor β3, and neuregulin 1. We demonstrate the derivation and culture of 34 hiPSC lines in B8 as well as the maintenance of pluripotency long term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing capacity for differentiation.

  • SFRP1 in Skin Tumor Initiation and Cancer Stem Cell Regulation with Potential Implications in Epithelial Cancers
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-09
    Raghava R. Sunkara; Rahul M. Sarate; Priyanka Setia; Sanket Shah; Sanjay Gupta; Pankaj Chaturvedi; Poonam Gera; Sanjeev K. Waghmare

    Wnt signaling is involved in the regulation of cancer stem cells (CSCs); however, the molecular mechanism involved is still obscure. SFRP1, a Wnt inhibitor, is downregulated in various human cancers; however, its role in tumor initiation and CSC regulation remains unexplored. Here, we used a skin carcinogenesis model, which showed early tumor initiation in Sfrp1−/− (Sfrp1 knockout) mice and increased tumorigenic potential of Sfrp1−/− CSCs. Expression profiling on Sfrp1−/− CSCs showed upregulation of genes involved in epithelial to mesenchymal transition, stemness, proliferation, and metastasis. Further, SOX-2 and SFRP1 expression was validated in human skin cutaneous squamous cell carcinoma, head and neck squamous cell carcinoma, and breast cancer. The data showed downregulation of SFRP1 and upregulation of SOX-2, establishing their inverse correlation. Importantly, we broadly uncover an inverse correlation of SFRP1 and SOX-2 in epithelial cancers that may be used as a potential prognostic marker in the management of cancer.

  • Recurrent Genetic Abnormalities in Human Pluripotent Stem Cells: Definition and Routine Detection in Culture Supernatant by Targeted Droplet Digital PCR
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-02
    Said Assou; Nicolas Girault; Mathilde Plinet; Julien Bouckenheimer; Caroline Sansac; Marion Combe; Joffrey Mianné; Chloé Bourguignon; Mathieu Fieldes; Engi Ahmed; Thérèse Commes; Anthony Boureux; Jean-Marc Lemaître; John De Vos
  • Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-02
    Tim Ahfeldt; Alban Ordureau; Christina Bell; Lily Sarrafha; Chicheng Sun; Silvia Piccinotti; Tobias Grass; Gustavo M. Parfitt; Joao A. Paulo; Fumiki Yanagawa; Takayuki Uozumi; Yasujiro Kiyota; J. Wade Harper; Lee L. Rubin

    Parkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.

  • Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-02
    Ling Ling; Xiafei Ren; Xue Cao; Afizah Binte Mohd Hassan; Sophia Mah; Padmapriya Sathiyanathan; Raymond A.A. Smith; Clarissa L.L. Tan; Michelle Eio; Rebekah M. Samsonraj; Andre J. van Wijnen; Michael Raghunath; Victor Nurcombe; James H. Hui; Simon M. Cool

    Human mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency.

  • A Stem Cell Reporter for Investigating Pluripotency and Self-Renewal in the Rat
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-02
    Stephen Meek; Jun Wei; Taeho Oh; Tom Watson; Jaime Olavarrieta; Linda Sutherland; Daniel F. Carlson; Angela Salzano; Tamir Chandra; Anagha Joshi; Tom Burdon

    Rat embryonic stem cells (rESCs) are capable of contributing to all differentiated tissues, including the germ line in chimeric animals, and represent a unique, authentic alternative to mouse embryonic stem cells for studying stem cell pluripotency and self-renewal. Here, we describe an EGFP reporter transgene that tracks expression of the benchmark naive pluripotency marker gene Rex1 (Zfp42) in the rat. Insertion of the EGFP reporter gene downstream of the Rex1 promoter disrupted Rex1 expression, but REX1-deficient rESCs and rats were viable and apparently normal, validating this targeted knockin transgene as a neutral reporter. The Rex1-EGFP gene responded to self-renewal/differentiation factors and validated the critical role of β-catenin/LEF1 signaling. The stem cell reporter also allowed the identification of functionally distinct sub-populations of cells within rESC cultures, thus demonstrating its utility in discriminating between cell states in rat stem cell cultures, as well as providing a tool for tracking Rex1 expression in the rat.

  • Transdifferentiation of Mouse Embryonic Fibroblasts into Dopaminergic Neurons Reactivates LINE-1 Repetitive Elements
    Stem. Cell Rep. (IF 5.499) Pub Date : 2020-01-02
    Francesco Della Valle; Manjula P. Thimma; Massimiliano Caiazzo; Salvatore Pulcrano; Mirko Celii; Sabir A. Adroub; Peng Liu; Gregorio Alanis-Lobato; Vania Broccoli; Valerio Orlando

    In mammals, LINE-1 (L1) retrotransposons constitute between 15% and 20% of the genome. Although only a few copies have retained the ability to retrotranspose, evidence in brain and differentiating pluripotent cells indicates that L1 retrotransposition occurs and creates mosaics in normal somatic tissues. The function of de novo insertions remains to be understood. The transdifferentiation of mouse embryonic fibroblasts to dopaminergic neuronal fate provides a suitable model for studying L1 dynamics in a defined genomic and unaltered epigenomic background. We found that L1 elements are specifically re-expressed and mobilized during the initial stages of reprogramming and that their insertions into specific acceptor loci coincides with higher chromatin accessibility and creation of new transcribed units. Those events accompany the maturation of neuronal committed cells. We conclude that L1 retrotransposition is a non-random process correlating with chromatin opening and lncRNA production that accompanies direct somatic cell reprogramming.

  • Analysis of Differentiation Protocols Defines a Common Pancreatic Progenitor Molecular Signature and Guides Refinement of Endocrine Differentiation
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-26
    Agata Wesolowska-Andersen; Rikke Rejnholdt Jensen; Marta Pérez Alcántara; Nicola L. Beer; Claire Duff; Vibe Nylander; Matthew Gosden; Lorna Witty; Rory Bowden; Mark I. McCarthy; Mattias Hansson; Anna L. Gloyn; Christian Honore

    Several distinct differentiation protocols for deriving pancreatic progenitors (PPs) from human pluripotent stem cells have been described, but it remains to be shown how similar the PPs are across protocols and how well they resemble their in vivo counterparts. Here, we evaluated three differentiation protocols, performed RNA and assay for transposase-accessible chromatin using sequencing on isolated PPs derived with these, and compared them with fetal human pancreas populations. This enabled us to define a shared transcriptional and epigenomic signature of the PPs, including several genes not previously implicated in pancreas development. Furthermore, we identified a significant and previously unappreciated cross-protocol variation of the PPs through multi-omics analysis and demonstrate how such information can be applied to refine differentiation protocols for derivation of insulin-producing beta-like cells. Together, our study highlights the importance of a detailed characterization of defined cell populations derived from distinct differentiation protocols and provides a valuable resource for exploring human pancreatic development.

  • LIN28B Impairs the Transition of hESC-Derived β Cells from the Juvenile to Adult State
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-26
    Xin Zhou; Gopika G. Nair; Holger A. Russ; Cassandra D. Belair; Mei-Lan Li; Mayya Shveygert; Matthias Hebrok; Robert Blelloch
  • iPSC-Derived Platelets Depleted of HLA Class I Are Inert to Anti-HLA Class I and Natural Killer Cell Immunity
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-26
    Daisuke Suzuki; Charlotte Flahou; Norihide Yoshikawa; Ieva Stirblyte; Yoshikazu Hayashi; Akira Sawaguchi; Marina Akasaka; Sou Nakamura; Natsumi Higashi; Huaigeng Xu; Takuya Matsumoto; Kosuke Fujio; Markus G. Manz; Akitsu Hotta; Hitoshi Takizawa; Koji Eto; Naoshi Sugimoto
  • Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-26
    Hitomi Matsunari; Masahito Watanabe; Koki Hasegawa; Ayuko Uchikura; Kazuaki Nakano; Kazuhiro Umeyama; Hideki Masaki; Sanae Hamanaka; Tomoyuki Yamaguchi; Masaki Nagaya; Ryuichi Nishinakamura; Hiromitsu Nakauchi; Hiroshi Nagashima
  • Histone H3 K4/9/27 Trimethylation Levels Affect Wound Healing and Stem Cell Dynamics in Adult Skin
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-19
    Sangjo Kang; Kylie Long; Sherry Wang; Aiko Sada; Tudorita Tumbar

    Epigenetic mechanisms controlling adult mammalian stem cell (SC) dynamics might be critical for tissue regeneration but are poorly understood. Mouse skin and hair follicle SCs (HFSCs) display reduced histone H3 K4me3, K9me3, and K27me3 methylation levels (hypomethylation) preceding hair growth. Chemical inhibition of relevant histone demethylases impairs subsequent differentiation and growth of HFs and delays wound healing. In wounding, this impairs epithelial cell differentiation and blood vessel recruitment, but not proliferation and fibroblast recruitment. With Aspm-CreER as a newfound inter-follicular epidermis lineage-labeling tool, and Lgr5-CreER for hair follicles, we demonstrate a reduced contribution of both lineages to wound healing after interfering with hypomethylation. Blocked hypomethylation increases BMP4 expression and selectively upregulates H3 K4me3 on the Bmp4 promoter, which may explain the effects on HFSC quiescence, hair cycle, and injury repair. Thus, transient hypomethylation of histone H3 K4/9/27me3 is essential for adult skin epithelial SC dynamics for proper tissue homeostasis and repair.

  • Conformal Coating of Stem Cell-Derived Islets for β Cell Replacement in Type 1 Diabetes
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-12
    Aaron A. Stock; Vita Manzoli; Teresa De Toni; Maria M. Abreu; Yeh-Chuin Poh; Lillian Ye; Adam Roose; Felicia W. Pagliuca; Chris Thanos; Camillo Ricordi; Alice A. Tomei

    The scarcity of donors and need for immunosuppression limit pancreatic islet transplantation to a few patients with labile type 1 diabetes. Transplantation of encapsulated stem cell-derived islets (SC islets) might extend the applicability of islet transplantation to a larger cohort of patients. Transplantation of conformal-coated islets into a confined well-vascularized site allows long-term diabetes reversal in fully MHC-mismatched diabetic mice without immunosuppression. Here, we demonstrated that human SC islets reaggregated from cryopreserved cells display glucose-stimulated insulin secretion in vitro. Importantly, we showed that conformally coated SC islets displayed comparable in vitro function with unencapsulated SC islets, with conformal coating permitting physiological insulin secretion. Transplantation of SC islets into the gonadal fat pad of diabetic NOD-scid mice revealed that both unencapsulated and conformal-coated SC islets could reverse diabetes and maintain human-level euglycemia for more than 80 days. Overall, these results provide support for further evaluation of safety and efficacy of conformal-coated SC islets in larger species.

  • Inducible Forward Programming of Human Pluripotent Stem Cells to Hemato-endothelial Progenitor Cells with Hematopoietic Progenitor Potential
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-12
    Lucas Lange; Dirk Hoffmann; Adrian Schwarzer; Teng-Cheong Ha; Friederike Philipp; Daniela Lenz; Michael Morgan; Axel Schambach

    Induced pluripotent stem cells (iPSCs) offer a promising platform to model early embryonic developmental processes, to create disease models that can be evaluated by drug screens as well as proof-of-concept experiments for regenerative medicine. However, generation of iPSC-derived hemato-endothelial and hematopoietic progenitor cells for these applications is challenging due to variable and limited cell numbers, which necessitates enormous up-scaling or development of demanding protocols. Here, we unravel the function of key transcriptional regulators SCL, LMO2, GATA2, and ETV2 (SLGE) on early hemato-endothelial specification and establish a fully inducible and stepwise hemato-endothelial forward programming system based on SLGE-regulated overexpression. Regulated induction of SLGE in stable SLGE-iPSC lines drives very efficient generation of large numbers of hemato-endothelial progenitor cells (CD144+/CD73–), which produce hematopoietic progenitor cells (CD45+/CD34+/CD38–/CD45RA−/CD90+/CD49f+) through a gradual process of endothelial-to-hematopoietic transition (EHT).

  • New Drugs for an Old Foe: Mycobacterium tuberculosis Meets PSC-Derived Macrophages
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-10
    Kathrin Haake, Nico Lachmann

    Infections with Mycobacterium tuberculosis (Mtb) are still among the top 10 causes of death worldwide, highlighting the utmost need for new forms of medical treatments. In this issue of Stem Cell Reports, Han et al. (2019) describe a technique to screen therapeutically active compounds targeting Mtb using pluripotent stem cell-derived macrophages.

  • Transcriptomically Guided Mesendoderm Induction of Human Pluripotent Stem Cells Using a Systematically Defined Culture Scheme
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-05
    Richard L. Carpenedo, Sarah Y. Kwon, R. Matthew Tanner, Julien Yockell-Lelièvre, Chandarong Choey, Carole Doré, Mirabelle Ho, Duncan J. Stewart, Theodore J. Perkins, William L. Stanford

    Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation.

  • Precision Health Resource of Control iPSC Lines for Versatile Multilineage Differentiation
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-05
    Matthew R. Hildebrandt, Miriam S. Reuter, Wei Wei, Naeimeh Tayebi, Jiajie Liu, Sazia Sharmin, Jaap Mulder, L. Stephen Lesperance, Patrick M. Brauer, Rebecca S.F. Mok, Caroline Kinnear, Alina Piekna, Asli Romm, Jennifer Howe, Peter Pasceri, Guoliang Meng, Matthew Rozycki, Deivid C. Rodrigues, James Ellis
  • Endothelial Cell-Selective Adhesion Molecule Contributes to the Development of Definitive Hematopoiesis in the Fetal Liver
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-12-05
    Tomoaki Ueda, Takafumi Yokota, Daisuke Okuzaki, Yoshihiro Uno, Tomoji Mashimo, Yoshiaki Kubota, Takao Sudo, Tomohiko Ishibashi, Yasuhiro Shingai, Yukiko Doi, Takayuki Ozawa, Ritsuko Nakai, Akira Tanimura, Michiko Ichii, Sachiko Ezoe, Hirohiko Shibayama, Kenji Oritani, Yuzuru Kanakura
  • Genetic Deletion of Hesx1 Promotes Exit from the Pluripotent State and Impairs Developmental Diapause
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-21
    Sara Pozzi, Sarah Bowling, John Apps, Joshua M. Brickman, Tristan A. Rodriguez, Juan Pedro Martinez-Barbera

    The role of the homeobox transcriptional repressor HESX1 in embryonic stem cells (ESCs) remains mostly unknown. Here, we show that Hesx1 is expressed in the preimplantation mouse embryo, where it is required during developmental diapause. Absence of Hesx1 leads to reduced expression of epiblast and primitive endoderm determinants and failure of diapaused embryos to resume embryonic development after implantation. Genetic deletion of Hesx1 impairs self-renewal and promotes differentiation toward epiblast by reducing the expression of pluripotency factors and decreasing the activity of LIF/STAT3 signaling. We reveal that Hesx1-deficient ESCs show elevated ERK pathway activation, resulting in accelerated differentiation toward primitive endoderm, which can be prevented by overexpression of Hesx1. Together, our data provide evidence for a novel role of Hesx1 in the control of self-renewal and maintenance of the undifferentiated state in ESCs and mouse embryos.

  • Human Neural Stem Cells Reinforce Hippocampal Synaptic Network and Rescue Cognitive Deficits in a Mouse Model of Alzheimer's Disease
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-21
    Ting Zhang, Wei Ke, Xuan Zhou, Yun Qian, Su Feng, Ran Wang, Guizhong Cui, Ran Tao, Wenke Guo, Yanhong Duan, Xiaobing Zhang, Xiaohua Cao, Yousheng Shu, Chunmei Yue, Naihe Jing

    Alzheimer's disease (AD) is characterized by memory impairments in its earliest clinical phase. The synaptic loss and dysfunction leading to failures of synaptic networks in AD brain directly cause cognitive deficits of patient. However, it remains unclear whether the synaptic networks in AD brain could be repaired. In this study, we generated functional human induced neural progenitor/stem cells (iNPCs) that had been transplanted into the hippocampus of immunodeficient wild-type and AD mice. The grafted human iNPCs efficiently differentiated into neurons that displayed long-term survival, progressively acquired mature membrane properties, formed graft-host synaptic connections with mouse neurons and functionally integrated into local synaptic circuits, which eventually reinforced and repaired the neural networks of host hippocampus. Consequently, AD mice with human iNPCs exhibited enhanced synaptic plasticity and improved cognitive abilities. Together, our results suggest that restoring synaptic failures by stem cells might provide new directions for the development of novel treatments for human AD.

  • SOX21 Ensures Rostral Forebrain Identity by Suppression of WNT8B during Neural Regionalization of Human Embryonic Stem Cells
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-21
    Zhuoqing Fang, Xinyuan Liu, Jing Wen, Fan Tang, Yang Zhou, Naihe Jing, Ying Jin

    The generation of brain region-specific progenitors from human embryonic stem cells (hESCs) is critical for their application. However, transcriptional regulation of neural regionalization in humans is poorly understood. Here, we applied a rostrocaudal patterning system from hESCs to dissect global transcriptional networks controlling early neural regionalization. We found that SOX21 is required for rostral forebrain fate specification. SOX21 knockout led to activation of Wnt signaling, resulting in caudalization of regional identity of rostral forebrain neural progenitor cells. Moreover, we identified WNT8B as a SOX21 direct target. Deletion of WNT8B or inhibition of Wnt signaling in SOX21 knockout neural progenitor cells restored rostral forebrain identity. Furthermore, SOX21 interacted with β-catenin, interfering with the binding of TCF4/β-catenin complex to the WNT8B enhancer. Collectively, these results unveil the unknown role of SOX21 and shed light on how a transcriptional factor modulates early neural regionalization through crosstalk with a key component of Wnt signaling.

  • Adult Human Dermal Progenitor Cell Transplantation Modulates the Functional Outcome of Split-Thickness Skin Xenografts
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-14
    Natacha A. Agabalyan, Holly D. Sparks, Samar Tarraf, Nicole L. Rosin, Katie Anker, Grace Yoon, Lindsay N. Burnett, Duncan Nickerson, Elena S. Di Martino, Vincent A. Gabriel, Jeff Biernaskie
  • The Ban on US Government Funding Research Using Human Fetal Tissues: How Does This Fit with the NIH Mission to Advance Medical Science for the Benefit of the Citizenry?
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-12
    Joseph M. McCune, Irving L. Weissman

    Some have argued that human fetal tissue research is unnecessary and/or immoral. Recently, the Trump administration has taken the drastic––and we believe misguided––step to effectively ban government-funded research on fetal tissue altogether. In this article, we show that entire lines of research and their clinical outcomes would not have progressed had fetal tissue been unavailable. We argue that this research has been carried out in a manner that is ethical and legal, and that it has provided knowledge that has saved lives, particularly those of pregnant women, their unborn fetuses, and newborns. We believe that those who support a ban on the use of fetal tissue are halting medical progress and therefore endangering the health and lives of many, and for this they should accept responsibility. At the very least, we challenge them to be true to their beliefs: if they wish to short-circuit a scientific process that has led to medical advances, they should pledge to not accept for themselves the health benefits that such advances provide.

  • Effects of Spaceflight on Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Structure and Function
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-07
    Alexa Wnorowski, Arun Sharma, Haodong Chen, Haodi Wu, Ning-Yi Shao, Nazish Sayed, Chun Liu, Stefanie Countryman, Louis S. Stodieck, Kathleen H. Rubins, Sean M. Wu, Peter H.U. Lee, Joseph C. Wu
  • Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-07
    Nicholas Bredenkamp, Jian Yang, James Clarke, Giuliano Giuseppe Stirparo, Ferdinand von Meyenn, Sabine Dietmann, Duncan Baker, Rosalind Drummond, Yongming Ren, Dongwei Li, Chuman Wu, Maria Rostovskaya, Sarah Eminli-Meissner, Austin Smith, Ge Guo

    In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.

  • Effective and Rapid Generation of Functional Neutrophils from Induced Pluripotent Stem Cells Using ETV2-Modified mRNA
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-07
    Vera S. Brok-Volchanskaya, David A. Bennin, Kran Suknuntha, Lucas C. Klemm, Anna Huttenlocher, Igor Slukvin
  • Environmental Elasticity Regulates Cell-type Specific RHOA Signaling and Neuritogenesis of Human Neurons
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-07
    Robert H. Nichol, Timothy S. Catlett, Massimo M. Onesto, Drew Hollender, Timothy M. Gómez
  • Reprogramming of Fibroblasts to Oligodendrocyte Progenitor-like Cells Using CRISPR/Cas9-Based Synthetic Transcription Factors
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-11-07
    Mantas Matjusaitis, Laura J. Wagstaff, Andrea Martella, Bart Baranowski, Carla Blin, Sabine Gogolok, Anna Williams, Steven M. Pollard
  • Transcriptional Profiling of Xenogeneic Transplants: Examining Human Pluripotent Stem Cell-Derived Grafts in the Rodent Brain
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-10-31
    Christopher R. Bye, Vanessa Penna, Isabelle R. de Luzy, Carlos W. Gantner, Cameron P.J. Hunt, Lachlan H. Thompson, Clare L. Parish

    Human pluripotent stem cells are a valuable resource for transplantation, yet our ability to profile xenografts is largely limited to low-throughput immunohistochemical analysis by difficulties in readily isolating grafts for transcriptomic and/or proteomic profiling. Here, we present a simple methodology utilizing differences in the RNA sequence between species to discriminate xenograft from host gene expression (using qPCR or RNA sequencing [RNA-seq]). To demonstrate the approach, we assessed grafts of undifferentiated human stem cells and neural progenitors in the rodent brain. Xenograft-specific qPCR provided sensitive detection of proliferative cells, and identified germ layer markers and appropriate neural maturation genes across the graft types. Xenograft-specific RNA-seq enabled profiling of the complete transcriptome and an unbiased characterization of graft composition. Such xenograft-specific profiling will be crucial for pre-clinical characterization of grafts and batch-testing of therapeutic cell preparations to ensure safety and functional predictability prior to translation.

  • Drug Discovery Platform Targeting M. tuberculosis with Human Embryonic Stem Cell-Derived Macrophages
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-10-31
    Hyo-Won Han, Hyang-Hee Seo, Hye-Yeong Jo, Hyeong-jun Han, Virgínia C.A. Falcão, Vincent Delorme, Jinyeong Heo, David Shum, Jang-Hoon Choi, Jin-Moo Lee, Seung Hun Lee, Hye-Ryeon Heo, Seok-Ho Hong, Mi-Hyun Park, Rajesh K. Thimmulappa, Jung-Hyun Kim
  • Patient-Specific iPSC Model of a Genetic Vascular Dementia Syndrome Reveals Failure of Mural Cells to Stabilize Capillary Structures
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-10-31
    Joseph Kelleher, Adam Dickinson, Stuart Cain, Yanhua Hu, Nicola Bates, Adam Harvey, Jianzhen Ren, Wenjun Zhang, Fiona C. Moreton, Keith W. Muir, Christopher Ward, Rhian M. Touyz, Pankaj Sharma, Qingbo Xu, Susan J. Kimber, Tao Wang

    CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most common form of genetic stroke and vascular dementia syndrome resulting from mutations in NOTCH3. To elucidate molecular mechanisms of the condition and identify drug targets, we established a patient-specific induced pluripotent stem cell (iPSC) model and demonstrated for the first time a failure of the patient iPSC-derived vascular mural cells (iPSC-MCs) in engaging and stabilizing endothelial capillary structures. The patient iPSC-MCs had reduced platelet-derived growth factor receptor β, decreased secretion of the angiogenic factor vascular endothelial growth factor (VEGF), were highly susceptible to apoptotic insults, and could induce apoptosis of adjacent endothelial cells. Supplementation of VEGF significantly rescued the capillary destabilization. Small interfering RNA knockdown of NOTCH3 in iPSC-MCs revealed a gain-of-function mechanism for the mutant NOTCH3. These disease mechanisms likely delay brain repair after stroke in CADASIL, contributing to the brain hypoperfusion and dementia in this condition, and will help to identify potential drug targets.

  • Closing the Mitochondrial Permeability Transition Pore in hiPSC-Derived Endothelial Cells Induces Glycocalyx Formation and Functional Maturation
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-10-31
    Gesa L. Tiemeier, Gangqi Wang, Sébastien J. Dumas, Wendy M.P.J. Sol, M. Cristina Avramut, Tobias Karakach, Valeria V. Orlova, Cathelijne W. van den Berg, Christine L. Mummery, Peter Carmeliet, Bernard M. van den Berg, Ton J. Rabelink
  • Notch Inhibition Enhances Cardiac Reprogramming by Increasing MEF2C Transcriptional Activity.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2017-03-07
    Maria Abad,Hisayuki Hashimoto,Huanyu Zhou,Maria Gabriela Morales,Beibei Chen,Rhonda Bassel-Duby,Eric N Olson

    Conversion of fibroblasts into functional cardiomyocytes represents a potential means of restoring cardiac function after myocardial infarction, but so far this process remains inefficient and little is known about its molecular mechanisms. Here we show that DAPT, a classical Notch inhibitor, enhances the conversion of mouse fibroblasts into induced cardiac-like myocytes by the transcription factors GATA4, HAND2, MEF2C, and TBX5. DAPT cooperates with AKT kinase to further augment this process, resulting in up to 70% conversion efficiency. Moreover, DAPT promotes the acquisition of specific cardiomyocyte features, substantially increasing calcium flux, sarcomere structure, and the number of spontaneously beating cells. Transcriptome analysis shows that DAPT induces genetic programs related to muscle development, differentiation, and excitation-contraction coupling. Mechanistically, DAPT increases binding of the transcription factor MEF2C to the promoter regions of cardiac structural genes. These findings provide mechanistic insights into the reprogramming process and may have important implications for cardiac regeneration therapies.

  • Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-10-10
    Caroline Halloin,Kristin Schwanke,Wiebke Löbel,Annika Franke,Monika Szepes,Santoshi Biswanath,Stephanie Wunderlich,Sylvia Merkert,Natalie Weber,Felix Osten,Jeanne de la Roche,Felix Polten,Kai Christoph Wollert,Theresia Kraft,Martin Fischer,Ulrich Martin,Ina Gruh,Henning Kempf,Robert Zweigerdt

  • Activation of IRF1 in Human Adipocytes Leads to Phenotypes Associated with Metabolic Disease.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2017-04-19
    Max Friesen,Raymond Camahort,Youn-Kyoung Lee,Fang Xia,Robert E Gerszten,Eugene P Rhee,Rahul C Deo,Chad A Cowan

    The striking rise of obesity-related metabolic disorders has focused attention on adipocytes as critical mediators of disease phenotypes. To better understand the role played by excess adipose in metabolic dysfunction it is crucial to decipher the transcriptional underpinnings of the low-grade adipose inflammation characteristic of diseases such as type 2 diabetes. Through employing a comparative transcriptomics approach, we identified IRF1 as differentially regulated between primary and in vitro-derived genetically matched adipocytes. This suggests a role as a mediator of adipocyte inflammatory phenotypes, similar to its function in other tissues. Utilizing adipose-derived mesenchymal progenitors we subsequently demonstrated that expression of IRF1 in adipocytes indeed contributes to upregulation of inflammatory processes, both in vitro and in vivo. This highlights IRF1's relevance to obesity-related inflammation and the resultant metabolic dysregulation.

  • Transmembrane Inhibitor of RICTOR/mTORC2 in Hematopoietic Progenitors.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2014-11-25
    Dongjun Lee,Stephen M Sykes,Demetrios Kalaitzidis,Andrew A Lane,Youmna Kfoury,Marc H G P Raaijmakers,Ying-Hua Wang,Scott A Armstrong,David T Scadden

    Central to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1) or are upstream and regulatory of it (mTORC2). Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2) negatively regulates mTORC2 enzymatic activity, reducing AKT(S473), PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL) model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway.

  • Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2014-07-30
    Raghavan Chinnadurai,Marco A Garcia,Yumiko Sakurai,Wilbur A Lam,Allan D Kirk,Jacques Galipeau,Ian B Copland

    Mesenchymal stromal cells have shown clinical promise; however, variations in treatment responses are an ongoing concern. We previously demonstrated that MSCs are functionally stunned after thawing. Here, we investigated whether this cryopreservation/thawing defect also impacts the postinfusion biodistribution properties of MSCs. Under both static and physiologic flow, compared with live MSCs in active culture, MSCs thawed from cryopreservation bound poorly to fibronectin (40% reduction) and human endothelial cells (80% reduction), respectively. This reduction correlated with a reduced cytoskeletal F-actin content in post-thaw MSCs (60% reduction). In vivo, live human MSCs could be detected in murine lung tissues for up to 24 hr, whereas thawed MSCs were undetectable. Similarly, live MSCs whose actin cytoskeleton was chemically disrupted were undetectable at 24 hr postinfusion. Our data suggest that post-thaw cryopreserved MSCs are distinct from live MSCs. This distinction could significantly affect the utility of MSCs as a cellular therapeutic.

  • The Developmental Stage of Adult Human Stem Cell-Derived Retinal Pigment Epithelium Cells Influences Transplant Efficacy for Vision Rescue.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2017-06-20
    Richard J Davis,Nazia M Alam,Cuiping Zhao,Claudia Müller,Janmeet S Saini,Timothy A Blenkinsop,Francesca Mazzoni,Melissa Campbell,Susan M Borden,Carol J Charniga,Patty L Lederman,Vanessa Aguilar,Michael Naimark,Michael Fiske,Nathan Boles,Sally Temple,Silvia C Finnemann,Glen T Prusky,Jeffrey H Stern

    Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.

  • A Subpopulation of Label-Retaining Cells of the Kidney Papilla Regenerates Injured Kidney Medullary Tubules.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2016-04-28
    Juan A Oliver,Rosemary V Sampogna,Sumreen Jalal,Qing-Yin Zhang,Alexander Dahan,Weiwei Wang,Tian Huai Shen,Qais Al-Awqati

    To determine whether adult kidney papillary label-retaining cells (pLRCs) are specialized precursors, we analyzed their transcription profile. Among genes overexpressed in pLRCs, we selected candidate genes to perform qPCR and immunodetection of their encoded proteins. We found that Zfyve27, which encodes protrudin, identified a subpopulation of pLRCs. With Zfyve27-CreERT2 transgenic and reporter mice we generated bitransgenic animals and performed cell-lineage analysis. Post tamoxifen, Zfyve27-CreERT2 marked cells preferentially located in the upper part of the papilla. These cells were low cycling and did not generate progeny even after long-term observation, thus they did not appear to contribute to kidney homeostasis. However, after kidney injury, but only if severe, they activated a program of proliferation, migration, and morphogenesis generating multiple and long tubular segments. Remarkably these regenerated tubules were located preferentially in the kidney medulla, indicating that repair of injury in the kidney is regionally specified. These results suggest that different parts of the kidney have different progenitor cell pools.

  • OSKM Induce Extraembryonic Endoderm Stem Cells in Parallel to Induced Pluripotent Stem Cells.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2016-03-08
    Anthony Parenti,Michael A Halbisen,Kai Wang,Keith Latham,Amy Ralston

    The reprogramming factors OCT4, SOX2, KLF4, and MYC (OSKM) can reactivate the pluripotency network in terminally differentiated cells, but also regulate expression of non-pluripotency genes in other contexts, such as the mouse primitive endoderm. The primitive endoderm is an extraembryonic lineage established in parallel to the pluripotent epiblast in the blastocyst, and is the progenitor pool for extraembryonic endoderm stem (XEN) cells. We show that OSKM induce expression of endodermal genes, leading to formation of induced XEN (iXEN) cells, which possess key properties of blastocyst-derived XEN cells, including morphology, transcription profile, self-renewal, and multipotency. Our data show that iXEN cells arise in parallel to induced pluripotent stem cells, indicating that OSKM drive cells to two distinct cell fates during reprogramming.

  • In Vitro Disease Modeling of Hermansky-Pudlak Syndrome Type 2 Using Human Induced Pluripotent Stem Cell-Derived Alveolar Organoids.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-07-11
    Yohei Korogi,Shimpei Gotoh,Satoshi Ikeo,Yuki Yamamoto,Naoyuki Sone,Koji Tamai,Satoshi Konishi,Tadao Nagasaki,Hisako Matsumoto,Isao Ito,Toyofumi F Chen-Yoshikawa,Hiroshi Date,Masatoshi Hagiwara,Isao Asaka,Akitsu Hotta,Michiaki Mishima,Toyohiro Hirai

  • Reprogramming Captures the Genetic and Tumorigenic Properties of Neurofibromatosis Type 1 Plexiform Neurofibromas.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-03-07
    Meritxell Carrió,Helena Mazuelas,Yvonne Richaud-Patin,Bernat Gel,Ernest Terribas,Imma Rosas,Senda Jimenez-Delgado,Josep Biayna,Leen Vendredy,Ignacio Blanco,Elisabeth Castellanos,Conxi Lázaro,Ángel Raya,Eduard Serra

  • Perspectives from the New Editor-in-Chief, Martin Pera.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2019-01-11
    Martin Pera

  • Transient Expression of WNT2 Promotes Somatic Cell Reprogramming by Inducing β-Catenin Nuclear Accumulation.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2016-05-24
    Mizuki Kimura,May Nakajima-Koyama,Joonseong Lee,Eisuke Nishida

    Treatment with several Wnt/β-catenin signaling pathway regulators can change the cellular reprogramming efficiency; however, the dynamics and role of endogenous Wnt/β-catenin signaling in reprogramming remain largely unanswered. Here we identify the upregulation of WNT2 and subsequent β-catenin nuclear accumulation as key events in reprogramming. Transient nuclear accumulation of β-catenin occurs early in MEF reprogramming. Wnt2 is strongly expressed in the early stage of reprogramming. Wnt2 knockdown suppresses the nuclear accumulation of β-catenin and reduces the reprogramming efficiency. WNT2 overexpression promotes β-catenin nuclear accumulation and enhances the reprogramming efficiency. WNT2 contributes to the promotion of cell proliferation. Experiments with several drugs that control the Wnt pathway also indicate the importance of β-catenin nuclear accumulation in reprogramming. Our findings reveal the role of WNT2/β-catenin signaling in reprogramming.

  • Using Zebrafish to Study Pathways that Regulate Hematopoietic Stem Cell Self-Renewal and Migration.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2017-06-08
    Avik Choudhuri,Eva M Fast,Leonard I Zon

    This perspective describes the usefulness of zebrafish as a model to study interaction of hematopoietic stem cells with the associated niche in vivo, explains how such interactions influence regeneration, migration, and clonality of HSCs, and defines their fate during differentiation.

  • Loss of Asxl1 Alters Self-Renewal and Cell Fate of Bone Marrow Stromal Cell, Leading to Bohring-Opitz-like Syndrome in Mice.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2016-05-31
    Peng Zhang,Caihong Xing,Steven D Rhodes,Yongzheng He,Kai Deng,Zhaomin Li,Fuhong He,Caiying Zhu,Lihn Nguyen,Yuan Zhou,Shi Chen,Khalid S Mohammad,Theresa A Guise,Omar Abdel-Wahab,Mingjiang Xu,Qian-Fei Wang,Feng-Chun Yang

    De novo ASXL1 mutations are found in patients with Bohring-Opitz syndrome, a disease with severe developmental defects and early childhood mortality. The underlying pathologic mechanisms remain largely unknown. Using Asxl1-targeted murine models, we found that Asxl1 global loss as well as conditional deletion in osteoblasts and their progenitors led to significant bone loss and a markedly decreased number of bone marrow stromal cells (BMSCs) compared with wild-type littermates. Asxl1(-/-) BMSCs displayed impaired self-renewal and skewed differentiation, away from osteoblasts and favoring adipocytes. RNA-sequencing analysis revealed altered expression of genes involved in cell proliferation, skeletal development, and morphogenesis. Furthermore, gene set enrichment analysis showed decreased expression of stem cell self-renewal gene signature, suggesting a role of Asxl1 in regulating the stemness of BMSCs. Importantly, re-introduction of Asxl1 normalized NANOG and OCT4 expression and restored the self-renewal capacity of Asxl1(-/-) BMSCs. Our study unveils a pivotal role of ASXL1 in the maintenance of BMSC functions and skeletal development.

  • Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2016-03-08
    Ge Guo,Ferdinand von Meyenn,Fatima Santos,Yaoyao Chen,Wolf Reik,Paul Bertone,Austin Smith,Jennifer Nichols

    Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.

  • Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2015-11-28
    Kristen J Brennand,M Carol Marchetto,Nissim Benvenisty,Oliver Brüstle,Allison Ebert,Juan Carlos Izpisua Belmonte,Ajamete Kaykas,Madeline A Lancaster,Frederick J Livesey,Michael J McConnell,Ronald D McKay,Eric M Morrow,Alysson R Muotri,David M Panchision,Lee L Rubin,Akira Sawa,Frank Soldner,Hongjun Song,Lorenz Studer,Sally Temple,Flora M Vaccarino,Jun Wu,Pierre Vanderhaeghen,Fred H Gage,Rudolf Jaenisch

    As a group, we met to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders. Although the convergence of findings between laboratories and patient cohorts provided us confidence and optimism that hiPSC-based platforms will inform future drug discovery efforts, a number of critical technical challenges remain. This opinion piece outlines our collective views on the current state of hiPSC-based disease modeling and discusses what we see to be the critical objectives that must be addressed collectively as a field.

  • Reprogramming of Polycomb-Mediated Gene Silencing in Embryonic Stem Cells by the miR-290 Family and the Methyltransferase Ash1l.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2015-11-10
    Chryssa Kanellopoulou,Timothy Gilpatrick,Gokhul Kilaru,Patrick Burr,Cuong K Nguyen,Aaron Morawski,Michael J Lenardo,Stefan A Muljo

    Members of the miR-290 family are the most abundantly expressed microRNAs (miRNAs) in mouse embryonic stem cells (ESCs). They regulate aspects of differentiation, pluripotency, and proliferation of ESCs, but the molecular program that they control has not been fully delineated. In the absence of Dicer, ESCs fail to express mature miR-290 miRNAs and have selective aberrant overexpression of Hoxa, Hoxb, Hoxc, and Hoxd genes essential for body plan patterning during embryogenesis, but they do not undergo a full differentiation program. Introduction of mature miR-291 into DCR(-/-) ESCs restores Hox gene silencing. This was attributed to the unexpected regulation of Polycomb-mediated gene targeting by miR-291. We identified the methyltransferase Ash1l as a pivotal target of miR-291 mediating this effect. Collectively, our data shed light on the role of Dicer in ESC homeostasis by revealing a facet of molecular regulation by the miR-290 family.

  • Successful Generation of Human Induced Pluripotent Stem Cell Lines from Blood Samples Held at Room Temperature for up to 48 hr.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2015-09-22
    Chukwuma A Agu,Filipa A C Soares,Alex Alderton,Minal Patel,Rizwan Ansari,Sharad Patel,Sally Forrest,Fengtang Yang,Jonathan Lineham,Ludovic Vallier,Christopher M Kirton

    The collection sites of human primary tissue samples and the receiving laboratories, where the human induced pluripotent stem cells (hIPSCs) are derived, are often not on the same site. Thus, the stability of samples prior to derivation constrains the distance between the collection site and the receiving laboratory. To investigate sample stability, we collected blood and held it at room temperature for 5, 24, or 48 hr before isolating peripheral blood mononuclear cells (PBMCs) and reprogramming into IPSCs. Additionally, PBMC samples at 5- and 48-hr time points were frozen in liquid nitrogen for 4 months and reprogrammed into IPSCs. hIPSC lines derived from all time points were pluripotent, displayed no marked difference in chromosomal aberration rates, and differentiated into three germ layers. Reprogramming efficiency at 24- and 48-hr time points was 3- and 10-fold lower, respectively, than at 5 hr; the freeze-thaw process of PBMCs resulted in no obvious change in reprogramming efficiency.

  • Comparative quantification of the surfaceome of human multipotent mesenchymal progenitor cells.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2015-02-17
    Rebecca J Holley,Guangping Tai,Andrew J K Williamson,Samuel Taylor,Stuart A Cain,Stephen M Richardson,Catherine L R Merry,Anthony D Whetton,Cay M Kielty,Ann E Canfield

    Mesenchymal progenitor cells have great therapeutic potential, yet incomplete characterization of their cell-surface interface limits their clinical exploitation. We have employed subcellular fractionation with quantitative discovery proteomics to define the cell-surface interface proteome of human bone marrow mesenchymal stromal/stem cells (MSCs) and human umbilical cord perivascular cells (HUCPVCs). We compared cell-surface-enriched fractions from MSCs and HUCPVCs (three donors each) with adult mesenchymal fibroblasts using eight-channel isobaric-tagging mass spectrometry, yielding relative quantification on >6,000 proteins with high confidence. This approach identified 186 upregulated mesenchymal progenitor biomarkers. Validation of 10 of these markers, including ROR2, EPHA2, and PLXNA2, confirmed upregulated expression in mesenchymal progenitor populations and distinct roles in progenitor cell proliferation, migration, and differentiation. Our approach has delivered a cell-surface proteome repository that now enables improved selection and characterization of human mesenchymal progenitor populations.

  • In vivo repopulating activity emerges at the onset of hematopoietic specification during embryonic stem cell differentiation.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2015-02-11
    Stella Pearson,Sara Cuvertino,Maud Fleury,Georges Lacaud,Valerie Kouskoff

    The generation of in vivo repopulating hematopoietic cells from in vitro differentiating embryonic stem cells has remained a long-standing challenge. To date, hematopoietic engraftment has mostly been achieved through the enforced expression of ectopic transcription factors. Here, we describe serum-free culture conditions that allow the generation of in vivo repopulating hematopoietic cells in the absence of ectopically expressed factors. We show that repopulating activity arises immediately upon the commitment of mesodermal precursors to the blood program, within the first wave of hematopoietic specification. We establish that the formation of these progenitors is extremely transient and exquisitely sensitive to the cytokine milieu. Our findings define the precise differentiating stage at which hematopoietic repopulating activity first appears in vitro, and suggest that during embryonic stem cell differentiation, all hematopoietic programs are unraveled simultaneously from the mesoderm in the absence of cues that restrict the coordinated emergence of each lineage as is normally observed during embryogenesis.

  • Genetic and functional diversity of propagating cells in glioblastoma.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2014-12-24
    Sara G M Piccirillo,Sue Colman,Nicola E Potter,Frederik W van Delft,Suzanne Lillis,Maria-Jose Carnicer,Lyndal Kearney,Colin Watts,Mel Greaves

    Glioblastoma (GBM) is a lethal malignancy whose clinical intransigence has been linked to extensive intraclonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies the implicit assumption that cancer stem cells or tumor-propagating cells are themselves genetically and functionally diverse. To test this, we screened primary GBM tumors by SNP array to identify copy number alterations (a minimum of three) that could be visualized in single cells by multicolor fluorescence in situ hybridization. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. Whole-exome sequencing and single-cell genetic analysis in one case revealed a more complex clonal structure. This proof-of-principle experiment revealed that subclones in each GBM had variable regenerative or stem cell activity, and highlighted genetic alterations associated with more competitive propagating activity in vivo.

  • Aberrant α-adrenergic hypertrophic response in cardiomyocytes from human induced pluripotent cells.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2014-11-25
    Gabor Földes,Elena Matsa,János Kriston-Vizi,Thomas Leja,Stefan Amisten,Ljudmila Kolker,Thusharika Kodagoda,Nazanin F Dolatshad,Maxime Mioulane,Karine Vauchez,Tamás Arányi,Robin Ketteler,Michael D Schneider,Chris Denning,Sian E Harding

    Cardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the α-adrenoceptor (αAR) agonist phenylephrine (PE) compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal α1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease.

  • BLIMP1 is required for postnatal epidermal homeostasis but does not define a sebaceous gland progenitor under steady-state conditions.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2014-11-02
    Kai Kretzschmar,Denny L Cottle,Giacomo Donati,Ming-Feng Chiang,Sven R Quist,Harald P Gollnick,Ken Natsuga,Kuo-I Lin,Fiona M Watt

    B-lymphocyte-induced nuclear maturation protein 1 (BLIMP1) was previously reported to define a sebaceous gland (SG) progenitor population in the epidermis. However, the recent identification of multiple stem cell populations in the hair follicle junctional zone has led us to re-evaluate its function. We show, in agreement with previous studies, that BLIMP1 is expressed by postmitotic, terminally differentiated epidermal cells within the SG, interfollicular epidermis, and hair follicle. Epidermal overexpression of c-Myc results in loss of BLIMP1(+) cells, an effect modulated by androgen signaling. Epidermal-specific deletion of Blimp1 causes multiple differentiation defects in the epidermis in addition to SG enlargement. In culture, BLIMP1(+) sebocytes have no greater clonogenic potential than BLIMP1(-) sebocytes. Finally, lineage-tracing experiments reveal that, under steady-state conditions, BLIMP1-expressing cells do not divide. Thus, rather than defining a sebocyte progenitor population, BLIMP1 functions in terminally differentiated cells to maintain homeostasis in multiple epidermal compartments.

  • Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-3-independent CD43(-) embryonic precursor.
    Stem. Cell Rep. (IF 5.499) Pub Date : 2014-09-23
    Stanislav Rybtsov,Antoniana Batsivari,Kateryna Bilotkach,Daria Paruzina,Jordi Senserrich,Oleg Nerushev,Alexander Medvinsky

    Definitive hematopoietic stem cells (HSCs) develop in the aorta gonad mesonephros (AGM) region in a stepwise manner. Type I pre-HSCs express CD41 but lack CD45 expression, which is subsequently upregulated in type II pre-HSCs prior to their maturation into definitive HSCs. Here, using ex vivo modeling of HSC development, we identify precursors of definitive HSCs in the trunk of the embryonic day 9.5 (E9.5) mouse embryo. These precursors, termed here pro-HSCs, are less mature than type I and II pre-HSCs. Although pro-HSCs are CD41(+), they lack the CD43 marker, which is gradually upregulated in the developing HSC lineage. We show that stem cell factor (SCF), but not interleukin-3 (IL-3), is a major effector of HSC maturation during E9-E10. This study extends further the previously established hierarchical organization of the developing HSC lineage and presents it as a differentially regulated four-step process and identifies additional targets that could facilitate the generation of transplantable HSCs from pluripotent cells for clinical needs.

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上海纽约大学William Glover