Small‐molecule–based generation of functional cardiomyocytes from human umbilical cord–derived induced pluripotent stem cells

2018 ◽  
Vol 120 (2) ◽  
pp. 1318-1327 ◽  
Author(s):  
Kai Hong Wu ◽  
Su Yun Wang ◽  
Qian Ru Xiao ◽  
Yu Yang ◽  
Ning Ping Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Umbilical Cord ◽  
Small Molecule ◽  
Pluripotent Stem Cells ◽  
Human Umbilical Cord ◽  
Induced Pluripotent

scholarly journals Umbilical Cord Tissue as a Source of Young Cells for the Derivation of Induced Pluripotent Stem Cells Using Non-Integrating Episomal Vectors and Feeder-Free Conditions

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 49
Author(s):  
Aisha Mohamed ◽  
Theresa Chow ◽  
Jennifer Whiteley ◽  
Amanda Fantin ◽  
Kersti Sorra ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Umbilical Cord ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Growth Media ◽  
Donor Cells ◽  
Episomal Vectors ◽  
Umbilical Cord Tissue ◽  
Induced Pluripotent

The clinical application of induced pluripotent stem cells (iPSC) needs to balance the use of an autologous source that would be a perfect match for the patient against any safety or efficacy issues that might arise with using cells from an older patient or donor. Drs. Takahashi and Yamanaka and the Office of Cellular and Tissue-based Products (PMDA), Japan, have had concerns over the existence of accumulated DNA mutations in the cells of older donors and the possibility of long-term negative effects. To mitigate the risk, they have chosen to partner with the Umbilical Cord (UC) banks in Japan to source allogeneic-matched donor cells. Production of iPSCs from UC blood cells (UCB) has been successful; however, reprogramming blood cells requires cell enrichment with columns or flow cytometry and specialized growth media. These requirements add to the cost of production and increase the manipulation of the cells, which complicates the regulatory approval process. Alternatively, umbilical cord tissue mesenchymal stromal cells (CT-MSCs) have the same advantage as UCB cells of being a source of young donor cells. Crucially, CT-MSCs are easier and less expensive to harvest and grow compared to UCB cells. Here, we demonstrate that CT-MSCs can be easily isolated without expensive enzymatic treatment or columns and reprogramed well using episomal vectors, which allow for the removal of the reprogramming factors after a few passages. Together the data indicates that CT-MSCs are a viable source of donor cells for the production of clinical-grade, patient matched iPSCs.

Genetic reprogramming of cord blood derived endothelial colony forming cells towards human induced pluripotent stem cells using episomal plasmids

2021 ◽  
pp. 1-18
Author(s):  
Osama Shahid ◽  
Sumbul Shamim ◽  
Jahan ara Ainuddin ◽  
Mohsin Wahid
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
University Hospital ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Time Duration ◽  
Endothelial Colony Forming Cells ◽  
Induced Pluripotent

Abstract Objective: This study aimed to isolate human umbilical cord blood derived endothelial colony forming cells (ECFCs) followed by their integration free reprogramming towards induced pluripotent stem cells (iPSCs) and molecular characterization of both cell types using multicolor flowcytometery and immunofluorescence respectively. Methods: The cord blood was collected from 37-39 weeks of gestational ages after C-section ex-utero from Dow University Hospital. The ECFCs isolated after ficoll based separation of cord blood mononuclear cells (CBMNCs) which on emergence characterized through flow cytometry and reprogrammed towards induced pluripotent stem cells (iPSCs) using episomal vectors, the iPSCs were characterized using immunofluorescence. The study was conducted at Stem Cells and Regenerative lab, Dow Research Institute of Biotechnology and Biomedical Sciences, Dow University of health sciences OJHA campus. The study time duration was about one year (October 2017-October 2018), study design was in vitro experimental. The sample size of the study was n=3.   Results: The isolated ECFCs were evaluated using Flowcytometery which showed positive expression for CD31, CD34, CD146 cell surface markers and negative for CD90. The successful reprogramming of ECFCs towards iPSCs was confirmed by immunofluorescence using OCT-4 which is considered to be a master regulator of pluripotency.  Conclusion: To the best of our knowledge this study was the first attempt to integration free reprogramming of cord blood derived endothelial colony forming cells towards induced pluripotent stem using Episomal plasmids. Cells that have been isolated from cord blood and those that have been reprogrammed both have potential therapeutic applications in regenerative medicine. Continuous...

216 EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS ANALOGOUS TO INNER CELL MASS-DERIVED LIF-DEPENDENT MOUSE EMBRYONIC STEM CELLS ESTABLISHED FROM THE DOMESTIC PIG, SUS SCROFA

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
B. P. Telugu ◽  
T. Ezashi ◽  
A. Alexenko ◽  
S. Lee ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Umbilical Cord ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Induced Pluripotent

Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The established piPSC are analogous to FGF2-dependent human (h) ESC and murine “epiblast stem cells,” and are likely to advance swine as a model in biomedical research. Here, we report for the first time, the establishment of LIF-dependent, so called naïve type pluripotent stem cells (1) from the inner cell mass (ICM) of porcine blastocysts by up-regulating the expression of KLF4 and POU5F1; and (2) from umbilical cord mesenchyme (Wharton's jelly) by transduction with OSKM factors and subsequent culture in the presence of LIF-based medium with inhibitors that substitute for low endogenous expression of c-MYC and KLF4 and promote pluripotency. The 2 compounds that have been used in this study are, CHIR99021 (CH), which substitutes c-MYC by inhibiting GSK3B and activating WNT signalling and Kenpaullone (KP), which inhibits both GSK3B and CDK1 and supplants KLF4 function. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression (tet-on) and are ‘floxed’ for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5.5 in vitro produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived stem cell lines are alkaline phosphatase-positive, resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile and expression of pluripotent markers, such as POU5F1, SOX2 and surface marker SSEA1. They are dependent on LIF signalling for maintenance of pluripotency, can be cultured over extended passage (>50) with no senescence. Of importance, the ICM-derived lines have been successful in their ability to form teratomas. The cells could be cultured in feeder free conditions on a synthetic matrix in the presence of chemically defined medium and can be coaxed to differentiate under xeno-free conditions. Currently, the piPSC lines are being investigated for their ability to give rise to teratomas and to produce a live offspring by nuclear transfer. Supported by Addgene Innovation Award, MO Life Sciences Board Grant 00022147 and NIH grant HD21896.

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
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