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.

small-molecules-03

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.

Cancer stem cells – friend or foe in the fight against cancer?

Today, following our series on Tumour Microenvironment (TME), and leading on from three recent publications, let’s discuss Cancer Stem Cells (CSCs).

CSCs are part of the TME, as they reside in niches where they can survive from immune surveillance, maintain plasticity and facilitate cancer metastasis (1). They can also circulate around the body. Which makes them so interesting. Their worse effect is that they can expand cancer to other organs in the body, thus helping metastasic. The good thing is that their early detection is a very powerful biomarker for cancer, allowing an early treatment. [Read more…]

A simple way to measure Elastin

Recently I reported about methods to measure:

Today, I invite you to take a closer look at another component of the extacellular matrix (ECM) – Elastin.

Visual representation of skin changes over a lifetime.

Visual representation of skin changes over a lifetime.

Elastin is a highly elastic protein in connective tissue and enables tissues in the body to resume their shape after contracting or stretching. Elastin helps skin to return to its original position when it is pinched or poked. During aging, elastin appearance decreases. The ELN gene encodes a protein which is rich in hydrophobic amino acids like glycine and proline, which form mobile hydrophobic regions bounded by crosslinks between lysine residues. A number of transcript variants encoding different isoforms are know for this gene. Together with the elastic microfibril (consisting of proteins such as microfibrillar-associated glycoproteins, fibrillin, fibullin, and the elastin receptor) elastin forms so called elastic fibers in the ECM of connective tissues.

Elastin and Diseases

Deletions and mutations in the ELN gene are associated with Supravalvular aortic stenosis (SVAS) and the autosomal dominant cutis laxa (or Chalazoderma or Dermatochalasia). Further elastin-related defects include Marfan syndrome, emphysema, atherosclerosis, Buschke-Ollendorff syndrome, Menkes syndrome, pseudoxanthoma elasticum, and Williams syndrome.

Detection of Elastin

Often elastin is detected with either ELISA or immunohistochemistry based methods. Our partner Biocolor developed a quantitative dye-binding method for the analysis of elastins released into tissue culture medium and extracted from biological materials (see the Fastin Flowchart in Fig.1)

Fastin flowchart

Fig. 1: Flowchart of the Fastin assay to measure elastin (click to view full size)

Which elastin forms can be measured by the Fastin Assay?

  • soluble tropoelastins
  • athyrogenic elastins
  • insoluble elastins (following solubilization to elastin polypeptides [α-elastin, κ-elastin])

    Fastin results

    Fig. 2: Extraction of elastin from mouse tissue by hot oxalic acid digestion. The extracts shown were pooled and expressed as μg elastin per 10 mg wet tissue.

The dye reagent (5,10,15,20-tetraphenyl-21 H, 23 H-porphine tetra-sulfonate, TPPS) binds to the ‘basic’ and ‘non-polar’ amino acid sequences found in mammalian elastins. The elastin-Dye complex can be read with a

microplate reader, with a suitable colour filter (absorbance peak of dye occurs at 513 nm).
Typical results of elastin measurements (in mouse tissue) are shown in Fig. 2.
Interested in measuring Elastin – or any other components of the extracellular matrix – in biological samples? Don’t hesitate to get in touch through the form below!

Pharmacological agents which alleviate Pain

Chronic pain of varying types including inflammatory pain, cancer pain and neuropathic pain is an increasing health problem for which there are inadequate therapeutic options.
Many new targets for analgesic therapeutics have been elucidated, these include TRP channels, the endocannabinoid system, sodium channels and various signaling pathways just to name a few.

Many new pharmacological agents which alleviate pain have been identified. In this post, I’d like to introduce some of them which have been recently released by Focus Biomolecules to support pain related research activities. [Read more…]

New in vitro cellular model for human Lipodystrophy

Lipodystrophies are disorders characterized by complete or selective loss of adipose tissue from various regions of the body. They might lead to severe metabolic disorders. The development of reliable cellular experimental models mimicking such diseases in vitro is extremely challenging (1). One of the main hurdle in the design of such in vitro cellular models is the access to reliable sources of well-qualified primary cells and the identification of optimal cell culture conditions.

[Read more…]

Enriched Tregs for a multitude of research applications

When it comes to the body’s natural defenses, is it possible to have “too much of a good thing”? Absolutely. To spare the host, the immune system needs to distinguish it from the real enemy –infectious agents. Autoimmune disorders occur when this recognition frays and the body’s own cells and tissues are damaged.

That’s where regulatory T cells, or Tregs, come in. [Read more…]

Factors affecting human islet cell quality

The islets of Langerhans are the regions of the pancreas that contain its endocrine (i.e., hormone-producing) cells. Discovered in 1869 by German pathological anatomist Paul Langerhans, the islets of Langerhans constitute approximately 1% to 2% of the mass of the pancreas. There are about one million islets distributed throughout the pancreas of a healthy adult human. Each is separated from the surrounding pancreatic tissue by a thin fibrous connective tissue capsule. The islets of Langerhans contain beta cells, which secrete insulin, and play a significant role in diabetes.

Islets are widely used for transplantation to restore beta cell function from diabetes, offering an alternative to a complete pancreas transplantation or an artificial pancreas. Because the beta cells in the islets of Langerhans are selectively destroyed by an autoimmune process in type 1 diabetes, islet transplantation is a means of restoring physiological beta cell function in patients with type 1 diabetes.

Human Islets for Research (HIR)™ are primary human islets processed from organ donor pancreases that have been approved for research but not for clinical transplantation of either the  pancreas or the isolated islets. HIR™ are obtained in a proprietary process of pancreas digestion and islet purification that results in uniformly high quality HIR™ for delivery to diabetes  investigators. Quality Control (QC) testing is routinely performed prior to release to assure uniform quality and function of these islets available for research. [Read more…]

Cellular models to study the Cardiac System (part II)

In a previous post, I introduced several models to study the cardiac human system. This first post introduced human aortic, brachiocephalic, carotid artery and coronary artery cells isolated by Cell Applications Inc. Here is the second part of this inventory of cellular models to study the human cardiac system, where I’ll be highlighting human internal thoracic artery, pulmonary artery, subclavian artery cells, cardiac fibroblasts and cardiomyocytes.

Later on, I’ll conclude this series by part III, referring to animal cellular models for studying the cardiac system. But let’s now concentrate on today’s topic! [Read more…]

Cellular models to study the cardiac System (part I)

In this post, I’d like to  introduce human aortic, brachiocephalic, carotid artery and coronary artery cells isolated by Cell Applications Inc. In a future post, I’ll be highlighting human internal thoracic artery, pulmonary artery, subclavian artery cells and cardiomyocytes (now published here).

After taking a look at several cell types as models for studying different aspects of cardiovascular functions and diseases, I’ll cover some recently published results highlighting the importance of securing your primary cells sourcing.

[Read more…]

Erythropoietin – active EPO for… hematologists!

EPO has a reputation with many non-scientists through bad press in the cycling/racing environment. From a researcher’s point of view, Erythropoietin or EPO is a 34 KDa glycoprotein known as the main player in erythropoiesis (red blood cell production). During fetal and perinatal period it’s predominantly produced in the liver, while in adults it is mainly released from interstitial fibroblasts in the kidney.

Besides its crucial role in erythropoiesis, EPO is also involved in wound healing and the brain’s response to neuronal injury. [Read more…]