Abstract 15547: Sfrp2 Mediates Ipsc to Cardiomyocyte Differentiation via Competing Actions on Wnts

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ying-chang Hsueh ◽  
Conrad Hodgkinson ◽  
Richard E Pratt ◽  
Victor J Dzau
Keyword(s):  
Ips Cells ◽  
Cardiomyocyte Differentiation ◽  
Blocking Antibody ◽  
Positive Effects ◽  
Wnt Proteins ◽  
Induced Pluripotent ◽  
Opposing Effects ◽  
Blocking Antibodies

Induced pluripotent stem (iPS) cells provide a novel avenue to investigate how precursors differentiate into cardiomyocytes. In this study we examined our hypothesis that iPS differentiation is regulated by a balance between cardiogenic and inhibitory Wnt proteins. Recently, we have discovered that iPS cells differentiate into cardiomyocytes in response to Sfrp2 (control: 0% cardiomyocytes; Sfrp2 treated: 25% cardiomyocytes. N=3. P<0.01). Since Sfrp2 mediates its effects by binding to Wnt proteins, we hypothesized that Sfrp2 promotes cardiomyocyte differentiation by binding to a Wnt that prevents iPS cells from differentiating into cardiomyocytes. To identify the inhibitory Wnt protein, we studied the effects of Wnt blocking antibodies on iPS and differentiation into cardiomyocytes. We observed that in control iPS cells or iPS cells incubated with a Wnt5 blocking antibody no cardiomyocytes appeared. In contrast, iPS cells incubated with a Wnt3a blocking antibody spontaneously and robustly differentiated into cardiomyocytes (40%, N=5, P<0.001). Co-immunoprecipitation experiments demonstrated that Sfrp2 binds to Wnt3a (N=3, P<0.001). Furthermore, Sfrp2 binding to Wnt3a inhibited b-catenin activation in iPS cells (80% active b-catenin in control cells; 10% active b-catenin in Sfrp2 and Wnt3a blocking antibody treated cells. N=3. P<0.05). To identify the cardiogenic Wnt, we again screened iPS differentiation following incubation with various Wnt blocking antibodies. Through this process, we found that the positive effects of Sfrp2 or Wnt3a blocking antibodies were completely lost when the iPS were co-cultured with Wnt11 blocking antibodies (Wnt3a blocking antibody alone: 40% cardiomyocytes; Wnt3a plus Wnt11 blocking antibody: 0% cardiomyocytes). Interestingly, in iPS cells incubated with Wnt11 blocking antibody, b-catenin was re-activated (control cells: 10% active b-catenin; cells incubated with Wnt11 blocking antibody: 60% active b-catenin. N=3. P<0.05). In summary, iPS cells do not spontaneously differentiate into cardiomyocytes because of opposing effects of Wnt3a and Wnt11. Sfrp2 promotes cardiomyocyte differentiation by binding to Wnt3a, allowing Wnt11 to induce iPS cells to differentiate into cardiomyocytes.

scholarly journals Cardiomyocyte differentiation from iPS cells is delayed following knockout of Bcl-2

2022 ◽  
Author(s):  
Tim Vervliet ◽  
Robin Duelen ◽  
lLewelyn H Roderick ◽  
Maurilio Sampaolesi
Keyword(s):  
Cell Death ◽  
Pluripotent Stem Cells ◽  
Direct Interaction ◽  
B Cell Lymphoma ◽  
Ips Cells ◽  
Cardiomyocyte Differentiation ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Induced Pluripotent

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) regulates a wide array of cellular functions involved in cell death, cell survival decisions and autophagy. Bcl-2 acts by both direct interaction with different components of the pathways involved and by intervening in intracellular Ca2+ signalling. The function of Bcl-2 is in turn regulated by post-translational modifications including phosphorylation at different sites by various kinases. Besides functions in cell death and apoptosis, Bcl-2 regulates cell differentiation processes, including of cardiomyocytes, although the signalling pathways involved are not fully elucidated. To further address the role of Bcl-2 during cardiomyocyte differentiation, we investigated the effect of its genetic knockout by CRISPR/Cas9 on the differentiation and functioning of human induced pluripotent stem cells to cardiomyocytes. Our results indicate that differentiation of iPS cells to cardiomyocytes is delayed by Bcl-2 KO, resulting in reduced size of spontaneously beating cells and reduced expression of cardiomyocyte Ca2+ toolkit and functionality. These data thus indicate that Bcl-2 an essential protein for cardiomyocyte generation.

scholarly journals The Effects of Tgfb1 and Csf3 on Chondrogenic Differentiation of iPS Cells in 2D and 3D Culture Environment

2021 ◽  
Vol 22 (6) ◽  
pp. 2978
Author(s):  
Chie-Hong Wang ◽  
Chun-Hao Tsai ◽  
Tsung-Li Lin ◽  
Shih-Ping Liu
Keyword(s):  
Cell Proliferation ◽  
Chondrogenic Differentiation ◽  
Expression Profiles ◽  
Es Cells ◽  
3D Culture ◽  
Ips Cells ◽  
3D Cultures ◽  
Positive Effects ◽  
Cartilaginous Matrix ◽  
Culture Environment

Mesenchymal stem (MS) cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells are known for their ability to differentiate into different lineages, including chondrocytes in culture. However, the existing protocol for chondrocyte differentiation is time consuming and labor intensive. To improve and simplify the differentiation strategy, we have explored the effects of interactions between growth factors (transforming growth factor β1 (Tgfb1) and colony stimulating factor 3 (Csf3), and culture environments (2D monolayer and 3D nanofiber scaffold) on chondrogenic differentiation. For this, we have examined cell morphologies, proliferation rates, viability, and gene expression profiles, and characterized the cartilaginous matrix formed in the chondrogenic cultures under different treatment regimens. Our data show that 3D cultures support higher proliferation rate than the 2D cultures. Tgfb1 promotes cell proliferation and viability in both types of culture, whereas Csf3 shows positive effects only in 3D cultures. Interestingly, our results indicate that the combined treatments of Tgfb1 and Csf3 do not affect cell proliferation and viability. The expression of cartilaginous matrix in different treatment groups indicates the presence of chondrocytes. We found that, at the end of differentiation stage 1, pluripotent markers were downregulated, while the mesodermal marker was upregulated. However, the expression of chondrogenic markers (col2a1 and aggrecan) was upregulated only in the 3D cultures. Here, we report an efficient, scalable, and convenient protocol for chondrogenic differentiation of iPS cells, and our data suggest that a 3D culture environment, combined with tgfb1 and csf3 treatment, promotes the chondrogenic differentiation.

scholarly journals Numerical Study of Fluid Dynamics in Suspension Culture of iPS Cells

2019 ◽  
Vol 17 (1) ◽  
pp. 73 ◽  
Author(s):  
Masaki Yano ◽  
Takuya Yamamoto ◽  
Yasunori Okano ◽  
Toshiyuki Kanamori ◽  
Mashiro Kino–oka
Keyword(s):  
Shear Stress ◽  
Suspension Culture ◽  
Numerical Study ◽  
Maximum Shear Stress ◽  
Ips Cells ◽  
Stirred Tanks ◽  
Average Shear Stress ◽  
Average Shear ◽  
Orbital Shaking ◽  
Induced Pluripotent

In a suspension culture of iPS cells, the shear stress generated during mixing is expected to promote differentiation of induced pluripotent stem (iPS) cells. The stress on the cells can be controlled by rotational rate and shape of impeller. However, it is difficult to optimize these operative parameters by experiments. Therefore, we have developed a numerical model to obtain the average and the maximum shear stress in two kinds of stirred tanks and an orbital shaking cylindrical container. The present results showed that the shear stress strongly depended on the type of mixing and lesser extent on the shape of the impeller. The average shear stress is larger in the shaking mode than that in the stirring mode. In contrast, the maximum shear stress is much smaller in the shaking than the stirring. These results suggest that stirring and shaking should be selectively used depending on the application

scholarly journals Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260852
Author(s):  
Meryem Ozgencil ◽  
Julian Barwell ◽  
Marc Tischkowitz ◽  
Louise Izatt ◽  
Ian Kesterton ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Repair ◽  
Ips Cells ◽  
Variants Of Uncertain Significance ◽  
Cellular Models ◽  
Pathogenic Variants ◽  
Damage Repair ◽  
Uncertain Significance ◽  
Induced Pluripotent ◽  
The Impact

Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

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