scholarly journals Generation of iPSCs by Nonintegrative RNA-Based Reprogramming Techniques: Benefits of Self-Replicating RNA versus Synthetic mRNA

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Heidrun Steinle ◽  
Marbod Weber ◽  
Andreas Behring ◽  
Ulrike Mau-Holzmann ◽  
Christian Schlensak ◽  
...  
Keyword(s):  
Disease Modeling ◽  
Somatic Cells ◽  
Patient Specific ◽  
Specific Marker ◽  
Messenger Rnas ◽  
Induced Pluripotent ◽  
Synthetic Mrna ◽  
Marker Detection

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is gaining in importance in the fields of regenerative medicine, tissue engineering, and disease modeling. Patient-specific iPSCs have as an unlimited cell source a tremendous potential for generating various types of autologous cells. For the future clinical applicability of these iPSC-derived cells, the generation of iPSCs via nongenome integrating methods and the efficient reprogramming of patients’ somatic cells are required. In this study, 2 different RNA-based footprint-free methods for the generation of iPSCs were compared: the use of synthetic modified messenger RNAs (mRNAs) or self-replicating RNAs (srRNAs) encoding the reprogramming factors and GFP. Using both RNA-based methods, integration-free iPSCs without genomic alterations were obtained. The pluripotency characteristics identified by specific marker detection and the in vitro and in vivo trilineage differentiation capacity were comparable. Moreover, the incorporation of a GFP encoding sequence into the srRNA enabled a direct and convenient monitoring of the reprogramming procedure and the successful detection of srRNA translation in the transfected cells. Nevertheless, the use of a single srRNA to induce pluripotency was less time consuming, faster, and more efficient than the daily transfection of cells with synthetic mRNAs. Therefore, we believe that the srRNA-based approach might be more appropriate and efficient for the reprogramming of different types of somatic cells for clinical applications.

scholarly journals Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell–Derived Endothelial Cells

2017 ◽  
Vol 37 (11) ◽  
pp. 2014-2025 ◽  
Author(s):  
Yang Lin ◽  
Chang-Hyun Gil ◽  
Mervin C. Yoder
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Induced Pluripotent ◽  
Human Ipsc

The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony–forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols.

scholarly journals The Promise of Human Induced Pluripotent Stem Cells in Dental Research

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Thekkeparambil Chandrabose Srijaya ◽  
Padmaja Jayaprasad Pradeep ◽  
Rosnah Binti Zain ◽  
Sabri Musa ◽  
Noor Hayaty Abu Kasim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Patient Specific ◽  
Dental Research ◽  
Cell Based Therapy ◽  
Induced Pluripotent ◽  
Disease Specific

Induced pluripotent stem cell-based therapy for treating genetic disorders has become an interesting field of research in recent years. However, there is a paucity of information regarding the applicability of induced pluripotent stem cells in dental research. Recent advances in the use of induced pluripotent stem cells have the potential for developing disease-specific iPSC linesin vitrofrom patients. Indeed, this has provided a perfect cell source for disease modeling and a better understanding of genetic aberrations, pathogenicity, and drug screening. In this paper, we will summarize the recent progress of the disease-specific iPSC development for various human diseases and try to evaluate the possibility of application of iPS technology in dentistry, including its capacity for reprogramming some genetic orodental diseases. In addition to the easy availability and suitability of dental stem cells, the approach of generating patient-specific pluripotent stem cells will undoubtedly benefit patients suffering from orodental disorders.

Abstract 518: In Vitro and in vivo Disease Modeling Using Patient Derived iPSCs to Characterize the Calcification Phenotype in Arterial Calcification Due to Deficiency of CD73

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Cynthia St. Hilaire ◽  
Hui Jin ◽  
Yuting Huang ◽  
Dan Yang ◽  
Alejandra Negro ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Dermal Fibroblasts ◽  
In Vivo Studies ◽  
Arterial Calcification ◽  
Patient Specific ◽  
And Control

Objective: The objective of this study was to develop a patient-specific induced pluripotent stem cell (iPSC)-based disease model to understand the process by which CD73-deficiency leads to vascular calcification in the disease, Arterial Calcification due to Deficiency of CD73 (ACDC). Approach & Results: ACDC is an autosomal recessive disease resulting from mutations in the gene encoding for CD73, which converts extracellular AMP to adenosine. CD73-deficiency manifests with tortuosity and vascular calcification of the medial layer of lower-extremity arteries, a pathology associated with diabetes and chronic kidney disease. We previously identified that dermal fibroblasts isolated from ACDC patients calcify in vitro, however in vivo studies of the vasculature are limited, as murine models of CD73 deficiency do not recapitulate the human disease phenotype. Thus, we created iPSCs from ACDC patients and control fibroblasts. ACDC and Control iPSCs form teratomas when injected in immune-compromised mice, however ACDC iPSC teratomas exhibit extensive calcifications. Control and ACDC iPSCs were differentiated down the mesenchymal lineage (MSC) and while there was no difference in chondrogenesis and adipogenesis, ACDC iMSCs underwent osteogenesis sooner than control iPSC, have higher activity of tissue-nonspecific alkaline phosphatase (TNAP), and lower levels of extracellular adenosine. During osteogenic simulation, TNAP activity in ACDC cells significantly increased adenosine levels, however, not to levels needed for functional compensatory stimulation of the adenosine receptors. Inhibition of TNAP with levimisole ablates this increase in adenosine. Treatment with an A2b adenosine receptor (AR) agonist drastically reduced TNAP activity in vitro, and calcification in ACDC teratomas, as did treatment with etidronate, which is currently being tested in a clinical trial on ACDC patients. Conclusions: These results illustrate a pro-osteogenic phenotype in CD73-deficient cells whereby TNAP activity attempts to compensate for CD73 deficiency, but subsequently induces calcification that can be reversed by activation of the A2bAR. The iPSC teratoma model may be used to screen other potential therapeutics for calcification disorders.

Abstract P445: A Combinatorial Approach Comprehensively Improves The Maturation Of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Olivia T Ly ◽  
Grace Brown ◽  
Hanna Chen ◽  
Liang Hong ◽  
Xinge Wang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Combinatorial Approach ◽  
Atrial Cardiomyocytes ◽  
Myocardial Substrate ◽  
Induced Pluripotent

Introduction: The limited success of pharmacological approaches to atrial fibrillation ( AF ) is due to limitations of in vitro and in vivo models and inaccessibility of human atrial tissue. Patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are a robust platform to model the heterogeneous myocardial substrate of AF, but their immaturity limits their fidelity. Objective: We hypothesized that a combinatorial approach of biochemical (triiodothyronine [ T3 ], insulin-like growth factor-1 [ IGF-1 ], and dexamethasone; collectively TID ), bioenergetic (fatty acids [ FA ]), and electrical stimulation ( ES ) will enhance electrophysiological ( EP ), structural, and metabolic maturity of iPSC- a CMs. Methods: We assessed maturation with whole cell patch clamping, calcium transients, immunofluorescence (IF), Seahorse Analyzer, contractility assay, RT-PCR, Western Blotting, and RNA sequencing (RNAseq). Using a time series with RNAseq we identified signaling pathways and transcriptional regulation that drive EP, structural, and metabolic atrial development and compared iPSC-aCM maturity with human aCMs (haCMs) obtained from the same patient. Results: TID+FA+ES significantly improved structural organization and cell morphology ( Fig. 1a ), enhanced membrane potential stability and improved depolarization ( Fig. 1b ), improved Ca 2+ kinetics with faster and increased Ca 2+ release from sarcoplasmic reticulum ( Fig. 1c ), and increased expression of Na + , Ca 2+ , and K + channels, markers of structural maturity, FA metabolism, and oxidative phosphorylation ( Fig. 1d ). There was no difference in each parameter between TID+FA+ES iPSC-aCMs and haCMs from the same patient. Conclusion: Our optimized, combinatorial TID+FA+ES approach markedly enhanced EP, structural, and metabolic maturity of human iPSC-aCMs, which will be useful for elucidating the genetic basis of AF developing precision drug therapies.

Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Anichavezhi Devendran ◽  
Rasheed Bailey ◽  
Sumanta Kar ◽  
Francesca Stillitano ◽  
Irene Turnbull ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Mononuclear Cells ◽  
Embryonic Stem ◽  
Model Systems ◽  
Patient Specific ◽  
Induced Pluripotent

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

Telomere Elongation in Dyskeratosis Congenita Induced Pluripotent Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 497-497
Author(s):  
Suneet Agarwal ◽  
Yuin-Han Loh ◽  
Erin M McLoughlin ◽  
Junjiu Huang ◽  
In-Hyun Park ◽  
...  
Keyword(s):  
Telomere Length ◽  
Autosomal Dominant ◽  
Somatic Cells ◽  
Ips Cells ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Patient Specific ◽  
Self Renewal ◽  
Induced Pluripotent

Abstract Abstract 497 Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer premature degeneration of multiple tissues. Bone marrow failure is the principal cause of mortality, and allogeneic stem cell transplantation is limited by increased treatment-related mortality. Somatic cells can be reprogrammed using defined genetic and chemical factors, yielding “induced pluripotent stem” (iPS) cell lines which have the capacity to differentiate into any tissue. Patient-specific iPS cells therefore hold promise as therapeutic agents and disease models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, and we have found that telomere length is increased in human iPS cells relative to the normal primary somatic cells from which they are derived. Here we investigated whether defects in telomerase function would limit derivation or self-renewal of iPS cells from patients with DC. We reprogrammed primary fibroblasts from patients with X-linked and autosomal dominant DC, caused by mutations in the genes encoding dyskerin and telomerase RNA component (TERC), respectively. We were able to establish multiple DC-specific iPS lines showing all hallmarks of pluripotency, including the formation of hematopoietic progenitors in vitro. Unexpectedly, DC-specific iPS cells were able to sustain continual proliferation in vitro, in contrast to the premature senescence displayed by the DC fibroblasts. Although early passage DC iPS cells had shorter telomeres than donor fibroblasts, we found that telomere length in DC iPS cells increased with continued passage in culture. To explain this finding, we discovered that steady state levels of TERC, which are critically limiting in several forms of DC, are upregulated in normal and DC iPS cells. We found that TERC upregulation is a feature of the pluripotent state, that the TERC locus is a target of pluripotency-associated transcription factors, and that transcriptional silencing accompanies a 3' deletion at the TERC locus in autosomal dominant DC. Our results demonstrate that reprogramming restores self-renewal capacity in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to enhance endogenous TERC expression may be feasible and therapeutically beneficial in DC patients. The studies demonstrate the value of patient-specific iPS cells for basic and translational discovery, and further the rationale for autologous iPS based cellular therapy of genetic hematologic disorders. Disclosures: Daley: MPM Capital: Consultancy; Solasia: Consultancy; Epizyme: Consultancy; iPierian: Consultancy, Equity Ownership.

scholarly journals Hypoxia as a Driving Force of Pluripotent Stem Cell Reprogramming and Differentiation to Endothelial Cells

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1614
Author(s):  
Paulina Podkalicka ◽  
Jacek Stępniewski ◽  
Olga Mucha ◽  
Neli Kachamakova-Trojanowska ◽  
Józef Dulak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Disease Modeling ◽  
Embryonic Stem ◽  
Hypoxic Conditions ◽  
Vascular Structures ◽  
Induced Pluripotent

Inadequate supply of oxygen (O2) is a hallmark of many diseases, in particular those related to the cardiovascular system. On the other hand, tissue hypoxia is an important factor regulating (normal) embryogenesis and differentiation of stem cells at the early stages of embryonic development. In culture, hypoxic conditions may facilitate the derivation of embryonic stem cells (ESCs) and the generation of induced pluripotent stem cells (iPSCs), which may serve as a valuable tool for disease modeling. Endothelial cells (ECs), multifunctional components of vascular structures, may be obtained from iPSCs and subsequently used in various (hypoxia-related) disease models to investigate vascular dysfunctions. Although iPSC-ECs demonstrated functionality in vitro and in vivo, ongoing studies are conducted to increase the efficiency of differentiation and to establish the most productive protocols for the application of patient-derived cells in clinics. In this review, we highlight recent discoveries on the role of hypoxia in the derivation of ESCs and the generation of iPSCs. We also summarize the existing protocols of hypoxia-driven differentiation of iPSCs toward ECs and discuss their possible applications in disease modeling and treatment of hypoxia-related disorders.

scholarly journals Running the full human developmental clock in interspecies chimeras using alternative human stem cells with expanded embryonic potential

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Justin Thomas ◽  
Ludovic Zimmerlin ◽  
Jeffrey S. Huo ◽  
Michael Considine ◽  
Leslie Cope ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adult Development ◽  
Disease Modeling ◽  
Scientific Evidence ◽  
Patient Specific ◽  
Human Stem Cells ◽  
Regulatory Oversight ◽  
Cell Therapies

AbstractHuman pluripotent stem cells (hPSCs) can generate specialized cell lineages that have great potential for regenerative therapies and disease modeling. However, the developmental stage of the lineages generated from conventional hPSC cultures in vitro are embryonic in phenotype, and may not possess the cellular maturity necessary for corrective regenerative function in vivo in adult recipients. Here, we present the scientific evidence for how adult human tissues could generate human–animal interspecific chimeras to solve this problem. First, we review the phenotypes of the embryonic lineages differentiated from conventional hPSC in vitro and through organoid technologies and compare their functional relevance to the tissues generated during normal human in utero fetal and adult development. We hypothesize that the developmental incongruence of embryo-stage hPSC-differentiated cells transplanted into a recipient adult host niche is an important mechanism ultimately limiting their utility in cell therapies and adult disease modeling. We propose that this developmental obstacle can be overcome with optimized interspecies chimeras that permit the generation of adult-staged, patient-specific whole organs within animal hosts with human-compatible gestational time-frames. We suggest that achieving this goal may ultimately have to await the derivation of alternative, primitive totipotent-like stem cells with improved embryonic chimera capacities. We review the scientific challenges of deriving alternative human stem cell states with expanded embryonic potential, outline a path forward for conducting this emerging research with appropriate ethical and regulatory oversight, and defend the case of why current federal funding restrictions on this important category of biomedical research should be liberalized.

scholarly journals Modeling Diamond Blackfan Anemia in Vivo Using Human Induced Pluripotent Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 359-359
Author(s):  
Sergei Doulatov ◽  
Linda T Vo ◽  
Elizabeth R Macari ◽  
Stephanie S Chou ◽  
Manav Gupta ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Erythroid Differentiation ◽  
Advisory Committees ◽  
Diamond Blackfan Anemia ◽  
Equity Ownership ◽  
Induced Pluripotent

Abstract Human induced pluripotent stem cells (iPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells has limited their use for modeling of hematological diseases. We previously reported a strategy to respecify lineage-restricted CD34+hematopoietic precursors derived from iPSCs into multilineage progenitors that can be expanded in vitro and transplanted in vivo. Five transcription factors, HOXA9, ERG, RORA, SOX4 and MYB, enable expansion and maintenance of primitive CD34+CD38- cells, and allow their differentiation upon transgene silencing. Respecified iPSC-derived hematopoietic progenitors give rise to robust short-term engraftment with myeloid and erythroid lineages. Notably, the erythrocytes undergo definitive maturation and hemoglobin switching to express β-globin in vivo. This system presents a useful platform for modeling hematological disorders due to its capacity to generate large numbers of engraftable disease cells for in vitro and in vivo screens. Congenital anemias, such as Diamond Blackfan anemia (DBA) represent a defined system for understanding red blood cell development and more common idiopathic anemias. To model this disease in vitro, we combined factor-induced respecification with stepwise erythroid maturation. We show that respecified iPSCs from DBA patients recapitulate the defect in erythroid differentiation. Consistent with clinical observations, early erythroblasts show impaired proliferation marked by increased apoptosis and p21 expression, while terminal maturation is unaffected. Interestingly, while early passage iPSC lines display a profound erythroid defect, continuous passage restores a near-normal capacity for erythroid differentiation. Furthermore, transplanted DBA iPSC-derived progenitors give rise to normal myeloid, but fail to generate erythroid, engraftment. This validates this system as a powerful tool for disease modeling and drug discovery. Mice transplanted with DBA iPSC-derived progenitors treated with conventional anemia drugs, such as dexamethasone, show a modest improvement in human erythroid output suggesting the need for novel treatments. Using high-throughput chemical screens, we have identified several signaling pathways which may be attractive therapeutic targets in DBA and other anemias. Further characterization is currently underway to better target defective erythropoiesis and dissect underlying disease mechanisms. Disclosures Zon: FATE Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Liver Organoids: Updates on Disease Modeling and Biomedical Applications

Biology ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 835
Author(s):  
Carmen Caiazza ◽  
Silvia Parisi ◽  
Massimiliano Caiazzo
Keyword(s):  
Stem Cells ◽  
Disease Modeling ◽  
Embryonic Stem ◽  
Low Complexity ◽  
In Vivo Models ◽  
Innovative Tool ◽  
Induced Pluripotent ◽  
Liver Organoids

Liver organoids are stem cell-derived 3D structures that are generated by liver differentiation signals in the presence of a supporting extracellular matrix. Liver organoids overcome low complexity grade of bidimensional culture and high costs of in vivo models thus representing a turning point for studying liver disease modeling. Liver organoids can be established from different sources as induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hepatoblasts and tissue-derived cells. This novel in vitro system represents an innovative tool to deeper understand the physiology and pathological mechanisms affecting the liver. In this review, we discuss the current advances in the field focusing on their application in modeling diseases, regenerative medicine and drug discovery.

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