Generation of Enterocyte-Like Cells with Pharmacokinetic Functions from Human Induced Pluripotent Stem Cells Using Small-Molecule Compounds

2015 ◽  
Vol 43 (4) ◽  
pp. 603-610 ◽  
Author(s):  
Takahiro Iwao ◽  
Nao Kodama ◽  
Yuki Kondo ◽  
Tomoki Kabeya ◽  
Katsunori Nakamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Small Molecule ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent

A Novel Chemical Approach to Expand Platelets Using Immortalized Megakaryocyte Progenitor Cells Derived from Human Induced Pluripotent Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3845-3845
Author(s):  
Ayako Otani ◽  
Tomo Koike ◽  
Natsuki Abe ◽  
Takanori Nakamura ◽  
Taito Nishino ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cells ◽  
Small Molecule ◽  
Pluripotent Stem Cells ◽  
Research Funding ◽  
Blue Staining ◽  
Platelet Production ◽  
Induced Pluripotent ◽  
Cell Stem Cell

Abstract Blood platelets can be obtained only by blood donation and reveal short-shelf life by the reason that they must be maintained with plasma at 20-24 degrees with shaking. These factors lead to shortage of donor platelets for clinical use. To overcome this issue, we developed a clinically applicable strategy for the derivation of functional platelets from human pluripotent stem cells (PSCs). We previously reported in vitro culture methods for producing functional platelets from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) (Takayama et al. Blood 2008, J Exp Med 2010). We also have established immortalized megakaryocyte progenitor cell lines (imMKCLs) with long-term expansion capability from hiPSC-derived hematopoietic progenitors with three defined factors, c-MYC, BMI1 and, BCL-XL (Nakamura et al. Cell Stem Cell, 2014). Although imMKCLs can be promising source of functional platelets for transfusion, further inventive efforts are needed to expand imMKCLs more efficiently towards clinical application. Thrombopoietin (TPO) is a cytokine initially identified as the primary regulator of megakaryocyte differentiation and platelet production. TPO is also an essential supplement for expanding platelets from imMKCLs. Recently, several nonpeptidyl small-molecule compounds have been developed to activate the TPO receptor, c-MPL and promote platelet production such as SB-497115 (Eltrombopag), an orally available drug for thrombocytopenia. Chemically synthesized c-MPL agonists reveal the advantage in terms of biological safety, low-immunogenicity or, low-cost manufacturing as compared to peptide-based ligands for platelet production. To obtain a c-MPL agonist that expands imMKCLs more efficiently and cost effectively than recombinant human TPO (rhTPO), we firstly screened small-molecule c-MPL agonists by evaluating its effects on platelet production from hiPSCs. Consequently, we identified “MK-001”, as the most potent compound that increases platelet productivity, as evidenced by the effects of MK-001 on the proliferation, differentiation, cell signaling, and platelet production from imMKCLs. We also studied the functionality of imMKCL-derived platelets. imMKCLs were cultured for 15 days with passage every 3 or 4 days with rhTPO in the presence of either rhTPO, Eltrombopag or MK-001 employing the same method as previously described (Nakamura et al. Cell Stem Cell, 2014). Total cell number was measured by Trypan Blue staining and automated cell counter. On day11, the number of total cells cultured with 200ng/mL of MK-001 was increased >1.5-fold compared with that of 50ng/mL of rhTPO (p<0.01). Subsequently, c-MYC, BMI1, and BCL-XL genes were turned off for inducing platelet yield. After another 4-days of culture, matured megakaryocytes and platelets were collected and analyzed by flow cytometer. Total number of CD41+CD42b+ platelets with MK-001 was increased >2-fold compared with that of rhTPO (p<0.001), whereas 1000ng/mL of Eltrombopag had little effect on platelet production. imMKCLs cultured with MK-001 contained a lot of large multinucleated cells and showed high levels of DNA content as well as those cultured with rhTPO. Intracellular phosphorylation analysis (BD Phosflow assay) revealed that MK-001 activated phosphorylation of components of three major TPO signaling pathways, JAK/STAT, MAPK, and PI3K/AKT within imMKCLs. PAC-1 bindings to platelets produced with MK-001, specific to an activated aIIbb3 integrin by adenosine 5-diphosphate (ADP), thrombin, or phorbol 12-myristate 13-acetate (PMA), were the same levels as those produced with rhTPO. These results indicated MK-001 promotes the production of functional platelets from imMKCLs more efficiently than rhTPO or Eltrombopag. In conclusion, the c-MPL agonist MK-001 could be applicable as an indispensable tool for expansion of platelets from hiPSCs with a combination of imMKCL system. Disclosures Nakauchi: Nissan Chemical Industries: Research Funding. Eto:Nissan Chemical Industries: Research Funding.

Purification of small molecule-induced cardiomyocytes from human induced pluripotent stem cells using a reporter system

2017 ◽  
Vol 232 (12) ◽  
pp. 3384-3395 ◽  
Author(s):  
Geun Hye Hwang ◽  
So Mi Park ◽  
Ho Jae Han ◽  
Joong Sun Kim ◽  
Seung Pil Yun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Small Molecule ◽  
Pluripotent Stem Cells ◽  
Reporter System ◽  
Induced Pluripotent

scholarly journals Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells

2012 ◽  
Vol 1 (2) ◽  
pp. 83-95 ◽  
Author(s):  
Yen Shun Chen ◽  
Rebecca A. Pelekanos ◽  
Rebecca L. Ellis ◽  
Rachel Horne ◽  
Ernst J. Wolvetang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Small Molecule ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent

scholarly journals A Small Molecule Modulating Monounsaturated Fatty Acids and Wnt Signaling Confers Maintenance to Induced Pluripotent Stem Cells Against Endodermal Differentiation

2021 ◽  
Author(s):  
Vahid Hosseini ◽  
Ashkan Kalantary-Charvadeh ◽  
Maryam Hajikarami ◽  
Parisa Fayyazpour ◽  
Reza Rahbarghazi ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Stem Cells ◽  
Wnt Signaling ◽  
Induced Pluripotent Stem Cells ◽  
Click Chemistry ◽  
Signaling Pathway ◽  
Small Molecule ◽  
Pluripotent Stem Cells ◽  
Endodermal Differentiation ◽  
Induced Pluripotent

Abstract Background: Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. This enzyme can orchestrate posttranslational modification of proteins involved in the development and differentiation of cells through the fatty acid acylation process. In this study, we evaluated whether a small molecule modulating unsaturated fatty acids influences early endodermal differentiation of induced pluripotent stem cells, using biochemical methods and immunostaining.Methods: The hiPSCs were cultured in an endoderm-inducing medium containing activin A and low defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The yield of three germ layers endoderm, mesoderm, and ectoderm, and the cell cycle analysis were assessed using flow cytometry. The expression of endoderm and pluripotency markers, as well as the expression of Wnt signaling pathway proteins, were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction.Results: The population of cells showing endoderm features was decreased at the end of differentiation when SCD1 was inhibited on the first day. Moreover, early SCD1 inhibition preserved hiPSCs properties without a shift toward mesoderm or ectoderm. Treatment of cells with SCD1 inhibitor only on the first day decreased the β-catenin gene expression and intensity of fluorescent emission in the click chemistry. These effects were effectively rescued by cotreatment with oleate. Late treatment at two subsequent days of endoderm induction induced no significant effect on endoderm-specific markers and fluorescent intensity. Reproducible results were also obtained with a human embryonic stem cell line. Conclusion: The small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring maintenance to hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in endoderm commitment of pluripotent stem cells may be eminent in disorders of endoderm-derived organs and dysregulated metabolism.

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