scholarly journals BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

2016 ◽  
Vol 113 (46) ◽  
pp. 13057-13062 ◽  
Author(s):  
Yohei Hayashi ◽  
Edward C. Hsiao ◽  
Salma Sami ◽  
Mariselle Lancero ◽  
Christopher R. Schlieve ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Fibrodysplasia Ossificans Progressiva ◽  
Expression Change ◽  
Major Barrier ◽  
Smad Signaling ◽  
Signaling Axis ◽  
Ipsc Generation ◽  
Id Genes ◽  
P16 Ink4a ◽  
Induced Pluripotent

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes.

scholarly journals Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1349
Author(s):  
Michael Stauske ◽  
Ignacio Rodriguez Polo ◽  
Wadim Haas ◽  
Debbra Yasemin Knorr ◽  
Thomas Borchert ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Cardiac Differentiation ◽  
Preclinical Development ◽  
Papio Anubis ◽  
Heart Repair ◽  
Large Numbers ◽  
Undifferentiated State ◽  
Advanced Therapy ◽  
Ipsc Generation ◽  
Induced Pluripotent

Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque (Macaca mulatta) iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (Papio anubis). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP.

scholarly journals Baboon induced pluripotent stem cell generation by piggyBac transposition of reprogramming factors

2019 ◽  
Vol 6 (2) ◽  
pp. 75-86 ◽  
Author(s):  
Ignacio Rodriguez-Polo ◽  
Michael Stauske ◽  
Alexander Becker ◽  
Iris Bartels ◽  
Ralf Dressel ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Large Body ◽  
Evolutionary Relationship ◽  
Induced Pluripotent Stem Cell ◽  
Papio Anubis ◽  
Cell Replacement ◽  
Ipsc Generation ◽  
Reprogramming Factors ◽  
Induced Pluripotent

Abstract. Clinical application of regenerative therapies using embryonic or induced pluripotent stem cells is within reach. Progress made during recent years has encouraged researchers to address remaining open questions in order to finally translate experimental cell replacement therapies into application in patients. To achieve this, studies in translationally relevant animal models are required to make the final step to the clinic. In this context, the baboon (Papio anubis) may represent a valuable nonhuman primate (NHP) model to test cell replacement therapies because of its close evolutionary relationship to humans and its large body size. In this study, we describe the reprogramming of adult baboon skin fibroblasts using the piggyBac transposon system. Via transposon-mediated overexpression of six reprogramming factors, we generated five baboon induced pluripotent stem cell (iPSC) lines. The iPSC lines were characterized with respect to alkaline phosphatase activity, pluripotency factor expression analysis, teratoma formation potential, and karyotype. Furthermore, after initial cocultivation with mouse embryonic fibroblasts, we were able to adapt iPSC lines to feeder-free conditions. In conclusion, we established a robust and efficient protocol for iPSC generation from adult baboon fibroblasts.

scholarly journals Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential

2018 ◽  
Vol 114 (14) ◽  
pp. 1828-1842 ◽  
Author(s):  
Alain van Mil ◽  
Geerthe Margriet Balk ◽  
Klaus Neef ◽  
Jan Willem Buikema ◽  
Folkert W Asselbergs ◽  
...  
Keyword(s):  
Heart Disease ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Cardiac Cells ◽  
Patient Specific ◽  
Specific Cell ◽  
Comprehensive Overview ◽  
Cellular Processes ◽  
Ipsc Generation ◽  
Induced Pluripotent

Abstract In the past few years, the use of specific cell types derived from induced pluripotent stem cells (iPSCs) has developed into a powerful approach to investigate the cellular pathophysiology of numerous diseases. Despite advances in therapy, heart disease continues to be one of the leading causes of death in the developed world. A major difficulty in unravelling the underlying cellular processes of heart disease is the extremely limited availability of viable human cardiac cells reflecting the pathological phenotype of the disease at various stages. Thus, the development of methods for directed differentiation of iPSCs to cardiomyocytes (iPSC-CMs) has provided an intriguing option for the generation of patient-specific cardiac cells. In this review, a comprehensive overview of the currently published iPSC-CM models for hereditary heart disease is compiled and analysed. Besides the major findings of individual studies, detailed methodological information on iPSC generation, iPSC-CM differentiation, characterization, and maturation is included. Both, current advances in the field and challenges yet to overcome emphasize the potential of using patient-derived cell models to mimic genetic cardiac diseases.

scholarly journals Generation of Functional Cardiomyocytes from Human Gastric Fibroblast-Derived Induced Pluripotent Stem Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1565
Author(s):  
Chih-Hsien Wu ◽  
Hsuan-Hwai Lin ◽  
Yi-Ying Wu ◽  
Yi-Lin Chiu ◽  
Li-Yen Huang ◽  
...  
Keyword(s):  
Coronary Artery ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent Stem Cell ◽  
Peptic Ulcers ◽  
Coronary Artery Diseases ◽  
The World ◽  
Ipsc Generation ◽  
Artery Disease ◽  
Induced Pluripotent

Coronary artery diseases are major problems of the world. Coronary artery disease patients frequently suffer from peptic ulcers when they receive aspirin treatment. For diagnostic and therapeutic purposes, the implementation of panendoscopy (PES) with biopsy is necessary. Some biopsy samples are wasted after the assay is completed. In the present study, we established a protocol for human gastric fibroblast isolation and induced pluripotent stem cell (iPSC) generation from gastric fibroblasts via PES with biopsy. We showed that these iPSCs can be differentiated into functional cardiomyocytes in vitro. To our knowledge, this is the first study to generate iPSCs from gastric fibroblasts in vitro.

scholarly journals Modeling the ACVR1R206H mutation in human skeletal muscle stem cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Emilie Barruet ◽  
Steven M Garcia ◽  
Jake Wu ◽  
Blanca M Morales ◽  
Stanley Tamaki ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cell ◽  
Fibrodysplasia Ossificans Progressiva ◽  
Regeneration Ability ◽  
Muscle Repair ◽  
Muscle Stem Cells ◽  
Marker Expression ◽  
Induced Pluripotent

Abnormalities in skeletal muscle repair can lead to poor function and complications such as scarring or heterotopic ossification (HO). Here, we use fibrodysplasia ossificans progressiva (FOP), a disease of progressive HO caused by ACVR1R206H (Activin receptor type-1 receptor) mutation, to elucidate how ACVR1 affects skeletal muscle repair. Rare and unique primary FOP human muscle stem cells (Hu-MuSCs) isolated from cadaveric skeletal muscle demonstrated increased ECM marker expression, showed skeletal muscle-specific impaired engraftment and regeneration ability. Human induced pluripotent stem cell (iPSC)-derived muscle stem/progenitor cells (iMPCs) single cell transcriptome analyses from FOP also revealed unusually increased ECM and osteogenic marker expression compared to control iMPCs. These results show that iMPCs can recapitulate many aspects of Hu-MuSCs for detailed in vitro study, that ACVR1 is a key regulator of Hu-MuSC function and skeletal muscle repair; and that ACVR1 activation in iMPCs or Hu-MuSCs may contribute to HO by changing the local tissue environment.

scholarly journals Expression of the retina-specific flippase, ABCA4, in epidermal keratinocytes

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 193 ◽  
Author(s):  
Luke A. Wiley ◽  
Emily E. Kaalberg ◽  
Jessica A. Penticoff ◽  
Robert F. Mullins ◽  
Edwin M. Stone ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Transmembrane Protein ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Epidermal Keratinocytes ◽  
Photoreceptor Outer Segment ◽  
Late Passage ◽  
Ipsc Generation ◽  
Scientific Expertise ◽  
Induced Pluripotent

ATP-binding cassette, sub-family A, member 4 (ABCA4) is a photoreceptor transmembrane protein that is responsible for flipping N-retinylidene-phosphatidylethanolamine, a key intermediate in the visual cycle, from the lumen to the cytoplasmic leaflet of photoreceptor outer segment disks. Mutations in ABCA4 cause a build-up of toxic retinoids resulting in a variety of retinal degenerative phenotypes, including Stargardt disease, cone-rod dystrophy and retinitis pigmentosa. Since many of the ABCA4 variants are rare and non-exomic, their pathogenicity is often difficult to demonstrate statistically. Given that the neural retina is inaccessible to molecular analysis in living patients, we use patient-specific induced pluripotent stem cell (iPSC)-derived retinal neurons to identify and model disease-causing mutations. Here we demonstrate that a truncated version of the retinal-specific transmembrane enzyme ABCA4 is expressed in epidermal keratinocytes and is required for cellular proliferation and viability at late passage. This finding is of great importance for labs that wish to investigate the pathophysiology of novel ABCA4-variants, without having to incur the added expense and scientific expertise associated with iPSC generation, culture and differentiation. Likewise, this finding is also important for those intending to generate iPSCs from patient specific keratinocytes, which can prove difficult when ABCA4 mutations are present.

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