New way for derivation / maintenance of naïve human PSCs

Newly added to the Stemolecule portfolio are three small molecules which support a new approach for the derivation and maintenance of naïve human pluripotent stem cells. These three newly identified small molecules, WH-4-023, SB590885 and IM-12, are all kinase inhibitors.

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ReproCELL is currently the only stem cell reagent company able to supply all components of the 5i/L/A (5 inhibitors/human LIF/Activin A) media supplement formulation needed for the derivation and maintenance of a “naïve” or ground-state of pluripotency of human cells as referenced in the October 2014 Cell Stem Cell paper by Theunissen et al. out of Dr. Rudolf Jaenisch’s Lab at MIT’s Whitehead Institute. The paper is titled “Systematic identification of culture conditions for induction and maintenance of naïve human pluripotency.”

Stemgent Stemolecule WH-4-023 (SRC Inhibitor)IM-12 Structure

Stemgent Stemolecule SB590885 (BRAF Inhibitor)

Stemgent Stemolecule IM-12 (GSK-3β Inhibitor)

 

The addition of these three new small molecules consolidates a 5i/L/A media supplement offering in one place. 5i/L/A media supplement consists of 5 kinase inhibitors; WH-4-023 SRC inhibitor, SB590885 BRAF inhibitor, IM-12 GSK-3β inhibitor, PD0325901 MEK inhibitor, Y27632 ROCK inhibitor, recombinant human leukemia inhibitory factor (LIF) and Activin A. The 5i/L/A media supplement allows for the derivation of and conversion of human PSCs to a naïve state of pluripotency hypothesized to be the human equivalent to the mouse ground state of pluripotency.

The Theunissen paper demonstrates the conversion of traditional human epiblastic pluripotent stem cells to a ground state of pluripotency thought to be equivalent to the traditional mouse embryonic stem cell state. The human equivalent to the mouse ground state of pluripotency is termed naïve. Naïve human PSCs differ from traditional human epiblastic ESCs in that human ESCs are FGF/BMP signaling dependent (equivalent to mouse epiblastic stem cells). While naïve PSCs are LIF/Stat3 signaling dependent (equivalent to traditional mouse ESCs). Both traditional mouse ESCs and naïve human PSCs demonstrate higher single cell viability when passaging. Likewise both cell types demonstrate enhanced proliferation when compared to epiblastic stem cells. These attributes make naïve human pluripotent stem cells a good choice for genetic manipulation and gene targeting applications.

Related products used in the paper that researchers may be interested in include: Human recombinant FGF-basic growth factor, CHIR99021 and doxycycline.

Formation of Mesenchymal Tissues in Alvetex® Scaffold

The Alvetex®Scaffold is a novel substrate that enables a solution for simple and routine 3D culture. It is composed of a highly porous polystyrene scaffold that has been engineered into a 200 micron thick membrane to enable entry of cells and efficient exchange of gases and solutes. Cells enter the fabric of the scaffold, retain their natural 3D structure, and form close 3D interactions with adjacent cells. Unlike conventional 2D culture, cells in Alvetex®Scaffold do not grow as monolayers and do not undergo the flattened shape transition that can result in aberrant changes to gene and protein expression and consequently cellular function.

Mesenchymal stem cells (MSCs) are adherent multipotent cells derived from tissue such as bone marrow and which possess the ability to differentiate in vitro into a number of tissue types including bone, cartilage and muscle [1].

In this post, we demonstrate that MSCs extracted from the bone marrow of adult rats can be successfully cultured in 3D in Alvetex®Scaffold and induced to differentiate into osteogenic and adipogenic derivatives more efficiently than their 2D counterparts. We also report bone formation and the production of extracellular matrix by MG63 cells which represent an established cell line derived from a human osteosarcoma. The data generated here is supported by peer-reviewed literature [2,3] and clearly shows that Alvetex®Scaffold promotes enhanced in vitro differentiation. [Read more…]

Genome Editing in Stem Cells: outsource or do-it-yourself?

Many researchers are facing a dilemma: they want to set up a CRISPR genome editing project but they can’t decide which cell line to use for genome editing. Even some of the most cost-effective genome editing cell line generation services like the one from GeneCopoeia will cost a few thousand euros, so picking the correct cell line and setting up the project correctly is very important. Researchers working in primary cells may find the idea of switching to an immortalized cell line a bit artificial. They dream of the possibility of editing the genome of a stem cell line which they can then differentiate into their tissue of choice as needed. [Read more…]

Don’t miss any neurotrophin!

Neurotrophins are important biomarkers in Neurobiology, including stem cell development to neural lineages. BDNF and NGF are the ones being studied in most cases. However, it has been found that neurotrophins do not act independently. Many publications have described the coordinated actions of 2 or more neurotrophins, especially in development, but also in the adult. [Read more…]

Characterization & Applications of Human Cardiomyocytes

ReproCardio 2 Human iPSC-derived cardiomyocytes have been extensively characterized for their functional responsiveness to the known cardiotoxic compounds. In all our tests, including assays of cardiotoxic compounds not correctly identified by the hERG assay, ReproCardio has generated results in complete agreement with clinical findings.

In this post, we’ll highlight the characterisation of ReproCardio 2 cardiomyocytes and illustrate the applications they are used for. [Read more…]

Mouse Embryonic Fibroblasts Troubleshooting Guide

Mouse Embryonic Fibroblasts (MEFs) are critical biological reagents in stem cell research. Used as “feeder cells” to maintain Embryonic Stem (ES) cells and induced Pluripotent Stem (iPS), the quality of MEFs strongly influences Stem cell culture conditions, and may react differently depending on culture conditions. In parallel, MEFs can also be reprogrammed into a variety of more mature differentiated functional cell types (cardiomyocytes, neurons…). During daily discussions with Stem cell biologists, I have been able to collect Frequently Asked Questions and technical tips related to MEFs.

[Read more…]

5 most popular posts in Stem Cells in 2014!

Following on from the 5 most popular posts in Cell Sourcing, today let’s look back at the 5 most visited blog posts in 2014 in the field of stem cells.

images#1 – Induced pluripotent neural stem cells in screening assay

Introducing Human Induced Pluripotent Stem Cells as a promising option for researchers creating in vitro models for the study of neurodegenerative diseases. We also highlight the benefits of using human iPS-derived neural cells vs. rodent primary neural cells.

 

4-Maintains Normal Karyotype after 20 Passages#2 – Cost-effective serum replacer for stem cell culture!

FBS is an inherently variable component of the cell culture system, so consistent performance demands extensive screening of manufactured lots. To circumvent this problem, companies began offering “stem cell qualified” serum… discover more in this post!

 

Comparison of standard reprogramming methodologies#3 – mRNA reprogramming system: fastest & safest!

Early reprogramming methods utilized retroviral (including lentiviral) vectors to introduce reprogramming-associated transcription factors to the target cells. While these pioneering experiments provided a fundamental proof-of-concept, these vectors required the integration of heritable viral DNA sequence into the host cell’s genome… Discover the benefits of a new mRNA reprogramming system!

 

EGF StemBeads vs soluble EGF in IF staining of E14 and E18 Cortical cells tebu-bio

#4 – Recent advances in Stem Cell culture: Encapsulated Stembeads growth factors

How can I optimise my media to cultivate Stem cells with stable and cost-effective Stem Cell Factors? Find all the answers in this post!

 

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#5 -Isogenic Knockout iPSC Lines for Modeling of CNS Disorders

Knockout iPSC lines that mimic loss of function of genes associated with various CNS disorders as novel tools for controlled experiments to decipher functionality of each disease‐associated gene, and thus the mechanism underlying the disease.

 

We hope you found these posts of interest, stay up to date in 2015 by following our future articles!

Cost-effective serum replacer for stem cell culture!

Traditionally, Fetal Bovine Serum (FBS) has been used as an additive to cell culture media for the in vitro growth of pluripotent stem cells. FBS is an inherently variable component of the cell culture system, so consistent performance demands extensive screening of manufactured lots. To circumvent this problem, companies began offering “stem cell qualified” serum. One issue with this solution is that the qualification is not always performed on a stem cell line relevant to the researcher’s own cell lines. Years later, researchers began using Life Technologies Knockout® Serum Replacement (KOSR) to grow and maintain undifferentiated human embryonic stem (ES) cells in culture. While KOSR has shown to be more stable than FBS and performs better in maintaining undifferentiated ES and induced pluripotent stem (iPS) cells, it is expensive and many researchers are today looking for a more cost effective media solution for their pluripotent stem cell culture. To address this need in the scientific community, GlobalStem now offers a more economical solution with their new PluriQ™ Serum Replacement. [Read more…]

Your neurons will forget Lipofectamine!

Mature cultures of neurons and other neural cells are extremely valuable for in vitro neurotoxicity studies and screening for agents that can slow, stop, or even reverse the course of neurodegenerative diseases.

The transfection of nucleic acids into neurons is essential for studying many aspects of neurobiology. However, neurons are among the most difficult cell types to transfect. They are very sensitive to culture conditions, presenting a particular challenge with regards to efficiencies. In addition to yielding low efficiencies, currently available cationic lipid reagents are often toxic to the cells, compromising post-transfection experimental results. While some viral mediated gene delivery systems have been shown to produce high efficiencies, they are very labor intensive and inconvenient for most researchers, along with the inherent danger and risk of provoking an immune response in the cell and/or interfering with the host genome. So what other solutions exist? This recently introduced alternative is well worth discovering… [Read more…]

Get 1.8 fold more cardiomyocytes with MesoFate medium

The analysis of mouse and human embryonic stem cell differentiation cultures has indicated the existence of a cardiovascular progenitor, one of the earliest stages of mesoderm specification to the cardiovascular lineage. In this post, we’ll look, step-by-step, at a new cardiovascular differentiation experimental procedure starting from human pluripotent Stem Cells.

[Read more…]