scholarly journals Induced pluripotent stem cells from subjects with Lesch-Nyhan disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Diane J. Sutcliffe ◽  
Ashok R. Dinasarapu ◽  
Jasper E. Visser ◽  
Joery den Hoed ◽  
Fatemeh Seifar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent Stem Cell ◽  
Shotgun Proteomics ◽  
Significant Heterogeneity ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Cell Layers ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Induced Pluripotent

AbstractLesch-Nyhan disease (LND) is an inherited disorder caused by pathogenic variants in the HPRT1 gene, which encodes the purine recycling enzyme hypoxanthine–guanine phosphoribosyltransferase (HGprt). We generated 6 induced pluripotent stem cell (iPSC) lines from 3 individuals with LND, along with 6 control lines from 3 normal individuals. All 12 lines had the characteristics of pluripotent stem cells, as assessed by immunostaining for pluripotency markers, expression of pluripotency genes, and differentiation into the 3 primary germ cell layers. Gene expression profiling with RNAseq demonstrated significant heterogeneity among the lines. Despite this heterogeneity, several anticipated abnormalities were readily detectable across all LND lines, including reduced HPRT1 mRNA. Several unexpected abnormalities were also consistently detectable across the LND lines, including decreases in FAR2P1 and increases in RNF39. Shotgun proteomics also demonstrated several expected abnormalities in the LND lines, such as absence of HGprt protein. The proteomics study also revealed several unexpected abnormalities across the LND lines, including increases in GNAO1 decreases in NSE4A. There was a good but partial correlation between abnormalities revealed by the RNAseq and proteomics methods. Finally, functional studies demonstrated LND lines had no HGprt enzyme activity and resistance to the toxic pro-drug 6-thioguanine. Intracellular purines in the LND lines were normal, but they did not recycle hypoxanthine. These cells provide a novel resource to reveal insights into the relevance of heterogeneity among iPSC lines and applications for modeling LND.

scholarly journals In Situ Expansion, Differentiation, and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid

2020 ◽  
Vol 127 (2) ◽  
pp. 207-224 ◽  
Author(s):  
Molly E. Kupfer ◽  
Wei-Han Lin ◽  
Vasanth Ravikumar ◽  
Kaiyan Qiu ◽  
Lu Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cells ◽  
Cardiac Muscle ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Pump Function ◽  
Induced Pluripotent

Rationale: One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes—a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. Objective: To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. Methods and Results: We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell–laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. Conclusions: This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.

scholarly journals Global trends in clinical trials involving pluripotent stem cells: a systematic multi-database analysis

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Julia Deinsberger ◽  
David Reisinger ◽  
Benedikt Weber
Keyword(s):  
Stem Cells ◽  
Clinical Trials ◽  
Pluripotent Stem Cells ◽  
Search Algorithm ◽  
Embryonic Stem ◽  
Induced Pluripotent Stem Cell ◽  
World Health ◽  
Clinical Use ◽  
Scientific Publications ◽  
Induced Pluripotent

Abstract Pluripotent stem cells (PSCs) hold great potential for novel therapeutic approaches to regenerate or replace functionally impaired tissues. Since the introduction of the induced pluripotent stem cell technology in 2006, the number of scientific publications on this topic has constantly been increasing. However, so far no therapy based on PSCs has found its way into routine clinical use. In this study, we examined research trends related to clinical trials involving PSCs based on data obtained from ClinicalTrials.gov, the ICTRP database from the World Health Organization, as well as from a search of all individual databases that are included in the ICTRP using a multistep search algorithm. Following a stringent inclusion/exclusion procedure 131 studies remained that could be classified as clinical trials involving PSCs. The magnitude of these studies (77.1%) was observational, which implies that no cells were transplanted into patients, and only a minority of studies (22.9%) were of an interventional study type. The number of clinical trials involving induced pluripotent stem cells (iPSCs, 74.8%) was substantially higher than the one involving embryonic stem cells (ESCs, 25.2%). However, the picture changes completely when focusing on interventional studies, where in the majority (73.3%) of cases ESCs were used. Interestingly, also the study duration was significantly shorter for interventional versus observational trials (p = 0.002). When focusing on the geographical study regions, it became obvious that the greatest part of all observational trials was performed in the USA (41.6%) and in France (16.8%), while the magnitude of interventional studies was performed in Asian countries (China 36.7%, Japan 13.3%, South Korea 10.0%) and in the field of ophthalmology. In summary, these results indicate that only a limited number of trials were focusing on the actual transplantation of PSCs into patients in a rather narrow field of diagnoses. The future will tell us, if the iPSC technology will ultimately overcome the current challenges and will finally make its way into routine clinical use.

scholarly journals Young at Heart: Pioneering Approaches to Model Nonischaemic Cardiomyopathy with Induced Pluripotent Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Aoife Gowran ◽  
Marco Rasponi ◽  
Roberta Visone ◽  
Patrizia Nigro ◽  
Gianluca L. Perrucci ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cells ◽  
Drug Screening ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Fibroblasts ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

A mere 9 years have passed since the revolutionary report describing the derivation of induced pluripotent stem cells from human fibroblasts and the first in-patient translational use of cells obtained from these stem cells has already been achieved. From the perspectives of clinicians and researchers alike, the promise of induced pluripotent stem cells is alluring if somewhat beguiling. It is now evident that this technology is nascent and many areas for refinement have been identified and need to be considered before induced pluripotent stem cells can be routinely used to stratify, treat and cure patients, and to faithfully model diseases for drug screening purposes. This review specifically addresses the pioneering approaches to improve induced pluripotent stem cell based models of nonischaemic cardiomyopathy.

scholarly journals A novel GNAS-mutated human induced pluripotent stem cell model for understanding GNAS-mutated tumors

Tumor Biology ◽  
2020 ◽  
Vol 42 (9) ◽  
pp. 101042832096258
Author(s):  
Katsuhito Watanabe ◽  
Takashi Nakamura ◽  
Shoko Onodera ◽  
Akiko Saito ◽  
Takahiko Shibahara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Epithelial Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Cell Model ◽  
Induced Pluripotent Stem Cell ◽  
Immunohistochemical Analysis ◽  
Induced Pluripotent

A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 ( GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS-mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNASR201H -mutated ( GNASR201H/+) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS-mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell–derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNASR201H-mutated induced pluripotent stem cells and GNASR201H-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNASR201H-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS-mutated diseases.

scholarly journals Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

2020 ◽  
Vol 6 (30) ◽  
pp. eaba7606 ◽  
Author(s):  
Kai Wang ◽  
Ruei-Zeng Lin ◽  
Xuechong Hong ◽  
Alex H. Ng ◽  
Chin Nien Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
High Efficiency ◽  
Induced Pluripotent Stem Cell ◽  
Vascular Networks ◽  
Temporal Modulation ◽  
Induced Pluripotent

Human induced pluripotent stem cell (h-iPSC)–derived endothelial cells (h-iECs) have become a valuable tool in regenerative medicine. However, current differentiation protocols remain inefficient and lack reliability. Here, we describe a method for rapid, consistent, and highly efficient generation of h-iECs. The protocol entails the delivery of modified mRNA encoding the transcription factor ETV2 at the intermediate mesodermal stage of differentiation. This approach reproducibly differentiated 13 diverse h-iPSC lines into h-iECs with exceedingly high efficiency. In contrast, standard differentiation methods that relied on endogenous ETV2 were inefficient and notably inconsistent. Our h-iECs were functionally competent in many respects, including the ability to form perfused vascular networks in vivo. Timely activation of ETV2 was critical, and bypassing the mesodermal stage produced putative h-iECs with reduced expansion potential and inability to form functional vessels. Our protocol has broad applications and could reliably provide an unlimited number of h-iECs for vascular therapies.

scholarly journals The mitochondrial protein CHCHD2 primes the differentiation potential of human induced pluripotent stem cells to neuroectodermal lineages

2016 ◽  
Vol 215 (2) ◽  
pp. 187-202 ◽  
Author(s):  
Lili Zhu ◽  
Aurora Gomez-Duran ◽  
Gabriele Saretzki ◽  
Shibo Jin ◽  
Katarzyna Tilgner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Mitochondrial Protein ◽  
Embryonic Stem ◽  
Induced Pluripotent Stem Cell ◽  
Clonal Variation ◽  
Differentiation Potential ◽  
Induced Pluripotent

Human induced pluripotent stem cell (hiPSC) utility is limited by variations in the ability of these cells to undergo lineage-specific differentiation. We have undertaken a transcriptional comparison of human embryonic stem cell (hESC) lines and hiPSC lines and have shown that hiPSCs are inferior in their ability to undergo neuroectodermal differentiation. Among the differentially expressed candidates between hESCs and hiPSCs, we identified a mitochondrial protein, CHCHD2, whose expression seems to correlate with neuroectodermal differentiation potential of pluripotent stem cells. We provide evidence that hiPSC variability with respect to CHCHD2 expression and differentiation potential is caused by clonal variation during the reprogramming process and that CHCHD2 primes neuroectodermal differentiation of hESCs and hiPSCs by binding and sequestering SMAD4 to the mitochondria, resulting in suppression of the activity of the TGFβ signaling pathway. Using CHCHD2 as a marker for assessing and comparing the hiPSC clonal and/or line differentiation potential provides a tool for large scale differentiation and hiPSC banking studies.

scholarly journals Different Responses to Drug Safety Screening Targets between Human Neonatal and Infantile Heart Tissue and Cardiac Bodies Derived from Human-Induced Pluripotent Stem Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-14
Author(s):  
Jan Trieschmann ◽  
Moritz Haustein ◽  
Annette Köster ◽  
Jürgen Hescheler ◽  
Konrad Brockmeier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Isometric Force ◽  
Induced Pluripotent Stem Cell ◽  
Myocardial Tissue ◽  
Force Frequency ◽  
Adrenergic Stimulation ◽  
Tissue Slices ◽  
Induced Pluripotent

Aims. Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) have become a promising tool in cardiovascular safety pharmacology. Immaturity of iPS-CMs remains an ongoing concern. We compared electrophysiological and contractile features of cardiac bodies (hiPS-CBs) derived from human-induced pluripotent stem cells and human neonatal and infantile myocardial slices relevant for drug screening. Methods and Results. Myocardial tissue slices were prepared from biopsies obtained from patients undergoing surgery for hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot (TOF). Electrophysiological features and response to Ik,r blockade as well as contractile properties were investigated using microelectrodes and isometric force measurements and were compared to hiPS-CBs. Both native myocardial tissue slices as well as hiPS-CBs showed action potential prolongation after Ik,r blockade, but early afterdepolarisations could be observed in native myocardial tissue slices only. The force-frequency relationship (FFR) varied at lower frequencies and was negative throughout at higher frequencies in hiPS-CBs. In contrast, native myocardial tissue slices exhibited positive, negative, and biphasic FFRs. In contrast to native myocardial tissue slices, hiPS-CBs failed to show an inotropic response to ß-adrenergic stimulation. Although all groups showed ß-adrenergic induced positive lusitropy, the effect was more pronounced in myocardial tissue slices. Conclusion. hiPS-CBs were able to reproduce AP prolongation after Ik,r blockade, but to a lesser extent compared to human neonatal and infantile myocardial tissue slices. Early afterdepolarisations could not be induced in hiPS-CBs. Contractile force was differently regulated by β-adrenergic stimulation in hiPS-CBs and the native myocardium. If used for cardiotoxicity screening, caution is warranted as hiPS-CBs might be less sensitive to pharmacologic targets compared to the native myocardium of neonates and infants.

Functional coculture of sympathetic neurons and cardiomyocytes derived from human-induced pluripotent stem cells

2020 ◽  
Vol 319 (5) ◽  
pp. H927-H937
Author(s):  
Annika Winbo ◽  
Suganeya Ramanan ◽  
Emily Eugster ◽  
Stefan Jovinge ◽  
Jonathan R. Skinner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Sympathetic Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

We present data on a functional coculture between human-induced pluripotent stem cell-derived sympathetic neurons and cardiomyocytes. Moreover, this study adds significantly to the available data on the electrophysiological function of human-induced pluripotent stem cell-derived sympathetic neurons.

scholarly journals Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

2015 ◽  
Vol 112 (35) ◽  
pp. 10950-10955 ◽  
Author(s):  
Julien Maruotti ◽  
Srinivas R. Sripathi ◽  
Kapil Bharti ◽  
John Fuller ◽  
Karl J. Wahlin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Small Molecule ◽  
Pluripotent Stem Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Induced Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Age Related Macular Degeneration ◽  
Age Related ◽  
Induced Pluripotent

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

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