scholarly journals Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes

2020 ◽  
Vol 318 (1) ◽  
pp. C163-C173
Author(s):  
Adrian G. Cadar ◽  
Tromondae K. Feaster ◽  
Kevin R. Bersell ◽  
Lili Wang ◽  
TingTing Hong ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Induced Pluripotent Stem Cell ◽  
Fluorescence Recovery ◽  
Cardiac Myofilament ◽  
Real Time Visualization ◽  
Induced Pluripotent

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

scholarly journals Major Histocompatibility Complex–Matched Arteries Have Similar Patency to Autologous Arteries in a Mauritian Cynomolgus Macaque Major Histocompatibility Complex–Defined Transplant Model

2019 ◽  
Vol 8 (15) ◽  
Author(s):  
John P. Maufort ◽  
Jacqueline S. Israel ◽  
Matthew E. Brown ◽  
Steve J. Kempton ◽  
Nicholas J. Albano ◽  
...  
Keyword(s):  
Major Histocompatibility Complex ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Small Diameter ◽  
Induced Pluripotent Stem Cell ◽  
Major Histocompatibility ◽  
Arterial Grafts ◽  
Histocompatibility Complex ◽  
Induced Pluripotent

Background Arterial bypass and interposition grafts are used routinely across multiple surgical subspecialties. Current options include both autologous and synthetic materials; however, each graft presents specific limitations. Engineering artificial small‐diameter arteries with vascular cells derived from induced pluripotent stem cells could provide a useful therapeutic solution. Banking induced pluripotent stem cells from rare individuals who are homozygous for human leukocyte antigen alleles has been proposed as a strategy to facilitate economy of scale while reducing the potential for rejection of induced pluripotent stem cell–derived transplanted tissues. Currently, there is no standardized model to study transplantation of small‐diameter arteries in major histocompatibility complex–defined backgrounds. Methods and Results In this study, we developed a limb‐sparing nonhuman primate model to study arterial allotransplantation in the absence of immunosuppression. Our model was used to compare degrees of major histocompatibility complex matching between arterial grafts and recipient animals with long‐term maintenance of patency and function. Unexpectedly, we (1) found that major histocompatibility complex partial haplomatched allografts perform as well as autologous control grafts; (2) detected little long‐term immune response in even completely major histocompatibility complex mismatched allografts; and (3) observed that arterial grafts become almost completely replaced over time with recipient cells. Conclusions Given these findings, induced pluripotent stem cell–derived tissue‐engineered blood vessels may prove to be promising and customizable grafts for future use by cardiac, vascular, and plastic surgeons.

scholarly journals Four new induced pluripotent stem cell lines produced from northern white rhinoceros with non-integrating reprogramming factors

2017 ◽  
Author(s):  
Marisa L. Korody ◽  
Cullen Pivaroff ◽  
Thomas D. Nguyen ◽  
Suzanne E. Peterson ◽  
Oliver A. Ryder ◽  
...  
Keyword(s):  
Endangered Species ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
White Rhinoceros ◽  
Reprogramming Factors ◽  
Stem Cell Lines ◽  
Induced Pluripotent ◽  
Conservation Of Endangered Species

AbstractSince we first published methods for generation of induced pluripotent stem cells from endangered species1, we have developed improved non-integrating methods for reprogramming the functionally extinct northern white rhinoceros, and generated iPSCs from four more individuals. Our work is part of a long-term plan for assisted reproduction for conservation of endangered species.

scholarly journals Induced pluripotent stem cells differentiation into cardiomyocytes by rotating suspension culture and in specific plates with microwells

2016 ◽  
Vol 71 (1) ◽  
pp. 46-50
Author(s):  
G. Budash ◽  
N. Bilko
Keyword(s):  
Stem Cells ◽  
Ascorbic Acid ◽  
Suspension Culture ◽  
Cell Line ◽  
Pluripotent Stem Cells ◽  
Fluorescent Protein ◽  
Induced Pluripotent Stem Cell ◽  
Embryoid Bodies ◽  
Induced Pluripotent Cells ◽  
Induced Pluripotent

In order to enhance the differentiation of induced pluripotent cells into cardiomyocytes, we compared two methods of embryoid bodies formation: differentiation in rotating suspension culture and formation of embryoid bodies from a predetermined number of pluripotent stem cells in microwells of AggreWell plates. We used transgenic murine induced pluripotent stem cell line AT25. Cell line expressed IRES-flanked enhanced green fluorescent protein (eGFP) under the control of cardiac alpha myosin heavy chain promoter (αMHC). We applied flow cytometry and fluorescence microscopyin order to test the efficiency of differentiation processes. Thus, differentiation of pluripotent stem cells in AggreWell plates without adding differentiation factors was more effective than differentiation in rotating suspension culture. However, we obtained the most amounts of cardiomyocytes on the 11-th day in rotating suspension culture with ascorbic acid, after we applied dorsomorfin, DMSO, ascorbic acid, G-CSF with the above-mentioned methods. The amount of GFP + cells was 2,71 ± 0,07%.

scholarly journals Long-term effects of human induced pluripotent stem cell-derived retinal cell transplantation in Pde6b knockout rats

2021 ◽  
Author(s):  
Jee Myung Yang ◽  
Sunho Chung ◽  
KyungA Yun ◽  
Bora Kim ◽  
Seongjun So ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Transplantation ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Histological Evaluation ◽  
Long Term Effects ◽  
Retinal Cells ◽  
Rpe Cells ◽  
Induced Pluripotent

AbstractRetinal degenerative disorders, including age-related macular degeneration and retinitis pigmentosa (RP), are characterized by the irreversible loss of photoreceptor cells and retinal pigment epithelial (RPE) cells; however, the long-term effect of implanting both human induced pluripotent stem cell (hiPSC)-derived RPE and photoreceptor for retinal regeneration has not yet been investigated. In this study, we evaluated the long-term effects of hiPSC-derived RPE and photoreceptor cell transplantation in Pde6b knockout rats to study RP; cells were injected into the subretinal space of the right eyes of rats before the appearance of signs of retinal degeneration at 2–3 weeks of age. Ten months after transplantation, we evaluated the cells using fundus photography, optical coherence tomography, and histological evaluation, and no abnormal cell proliferation was observed. A relatively large number of transplanted cells persisted during the first 4 months; subsequently, the number of these cells decreased gradually. Notably, immunohistochemical analysis revealed that the hiPSC-derived retinal cells showed characteristics of both RPE cells and photoreceptors of human origin after transplantation. Functional analysis of vision by scotopic electroretinogram revealed significant preservation of vision after transplantation. Our study suggests that the transplantation of hiPSC-derived retinal cells, including RPE cells and photoreceptors, has a potential therapeutic effect against irreversible retinal degenerative diseases.

scholarly journals Clonal genetic and hematopoietic heterogeneity among human-induced pluripotent stem cell lines

Blood ◽  
2013 ◽  
Vol 122 (12) ◽  
pp. 2047-2051 ◽  
Author(s):  
Jason A. Mills ◽  
Kai Wang ◽  
Prasuna Paluru ◽  
Lei Ying ◽  
Lin Lu ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Induced Pluripotent Stem Cell ◽  
Specific Gene ◽  
Rare Cnvs ◽  
Developmental Potential ◽  
Hematopoietic Development ◽  
Key Points ◽  
Stem Cell Lines ◽  
Induced Pluripotent ◽  
Selection Of

Key Points Normal induced pluripotent stem cells exhibit donor-specific gene expression signatures and the capacity for hematopoietic development. CNVs acquired during reprogramming or selection of rare CNVs present in the starting cell population may alter iPSC developmental potential.

scholarly journals Development of Multilayer Mesenchymal Stem Cell Cell Sheets

2021 ◽  
Vol 1 (1) ◽  
pp. 4-24
Author(s):  
Jun Ochiai ◽  
Yutaka Niihara ◽  
Joan Oliva
Keyword(s):  
Stem Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Cell Sheets ◽  
Gene Therapies ◽  
The Past ◽  
Long Term Effect ◽  
Induced Pluripotent ◽  
New Guidelines

Cell and gene therapies have been developing dramatically over the past decade. To face and adapt to the development of these new therapies, the Food and Drug Administration (FDA) wrote and updated new guidelines from 2016 and keep updating them. Mesenchymal stem cells (MSCs) are the most used cells for treatment, far ahead from the induced pluripotent stem cells (iPSCs), based on registered clinical trials at clinicaltrials.gov. They are widely used because of their differentiation capacity and their anti-inflammatory properties, but some controversies still require clear answers. Additional studies are needed to determine the dosage, the number, and the route of injections (location and transplantation method), and if allogenic MSCs are safe compared to autologous MSC injection, including their long-term effect. In this review, we summarize the research our company is conducting with the adipose stromal cells in engineering cell sheets and their potential application.

scholarly journals Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production

2021 ◽  
Vol 22 (15) ◽  
pp. 8224
Author(s):  
Linda Krisch ◽  
Gabriele Brachtl ◽  
Sarah Hochmann ◽  
André Cronemberger Andrade ◽  
Michaela Oeller ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Iterative Process ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Mesoderm Induction ◽  
Related Protein ◽  
Media Composition ◽  
Platelet Production ◽  
Induced Pluripotent ◽  
Related Proteins

Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet’s coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.

scholarly journals Induced Pluripotent Stem Cells for Duchenne Muscular Dystrophy Modeling and Therapy

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 253 ◽  
Author(s):  
Lubos Danisovic ◽  
Martina Culenova ◽  
Maria Csobonyeiova
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Reprogramming ◽  
Progressive Degeneration ◽  
Cardiac Muscles ◽  
Dmd Gene ◽  
Induced Pluripotent

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, caused by mutation of the DMD gene which encodes the protein dystrophin. This dystrophin defect leads to the progressive degeneration of skeletal and cardiac muscles. Currently, there is no effective therapy for this disorder. However, the technology of cell reprogramming, with subsequent controlled differentiation to skeletal muscle cells or cardiomyocytes, may provide a unique tool for the study, modeling, and treatment of Duchenne muscular dystrophy. In the present review, we describe current methods of induced pluripotent stem cell generation and discuss their implications for the study, modeling, and development of cell-based therapies for Duchenne muscular dystrophy.

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