Human induced pluripotent stem (iPS) cells and cells differentiated from iPS cells have widely been used for in vivo models human disease progression. Jason Meyer, of Indiana University Purdue University Indianapolis, uses iPS cell-derived models to study retinogenesis and retinal disease. Two recent papers from his lab highlight the benefits of using Stemgent’s RNA reprogramming technology to enable robust differentiation of iPS cells to the retinal lineage (1, 2). RNA reprogramming technology was chosen in order for these studies to ensure that no vestiges of the reprogramming vectors were retained by the cells or integrated into the genome.
The introduction of transgenes into stem cells has shown to be a valuable experimental technique for studying stem cell biology. Transfecting stem cells without inhibiting cell viability and cell growth has shown to be difficult. DNA-In® Stem Transfection Reagent offers a simple, robust and reproducible method for delivering DNA into a wide range of stem cells, including neural stem cells. Formulated and optimized specifically for embryonic and adult stem cells, DNA-In® Stem is a new-generation transfection reagent that enables high efficiency transfection while maintaining maximum cell viability and cell growth.
In this post, I invite you to discover the benefits of using DNA-In® Stem Transfection Reagent vs. other reagents. A lot of pictures and graphs rather than long descriptions! Last but not least, DNA-In® Stem Transfection Reagent is less expensive compared to Lipofectamine reagents… [Read more…]
Hyaluronic acid (Hyaluronan) belongs to the group of glycosaminoglycans (GAGs), but unlike other GAGs like Heparan sulfate, Chondroitin sulfate or Keratan sulfate, it cannot be found as a proteoglycan. It represents a non-sulfated polysaccharide consisting of alternating residues of D-glucuronic acid and N-acetylglucosamine (Fig 1). In the human umbilical cord and synovial fluid, the size of HA is reported to be about 3,000,000 Da (a flexible chain of 4000 disaccharide units).
HA is one of the major components of the extracellular matrix and it absorbs high amounts of water thus giving tissues the ability to resist compression. Furthermore, Hyaluronan contributes significantly to cell proliferation and cell migration. As HA levels often correlate with malignancy and poor prognosis of certain cancers, it can be used as a tumor marker. As Hyaluronan plays a role in skin healing, it’s a widely used ingredient of skin-care products. [Read more…]
Finding new biomarkers for diagnosis, prognosis or prediction is a hot area in clinical & translational research. Three recent publications are a good example of this. [Read more…]
The inadequacy of animal models to predict human biology in the drug development process is becoming increasingly clear, due to species differences in uptake and metabolism at both cellular and organ levels.
As a result, there is a need for more human model systems to be incorporated earlier in research and development.
Innovative concepts such as “body on a chip” have been introduced, but the complexity and miniaturization of many of the formats has limited applicability on a commercial scale.
SciKon is developing tools that better recapitulate biological systems in bench-top cell culture formats, which are amenable to mass manufacturing (introduced recently in the post Cell Signaling isn’t static…your cell culture shouldn’t be either!). [Read more…]
Lipotransfers are ideal for restorative surgery, but retention is a problem. In a recent study, PRP grade concentrated platelets were used for a study examining how platelet-rich plasma helps to enhance fat graft survival.
In this age of crash diets and liposuction, it might sound surprising that some people receive fat transplants. But seriously, fat grafting is widely used and valued as a feasible method for addressing moderate defects caused by injuries, surgical removal of tumours, and congenital deficiencies. Fat grafting is safe and has the look and feel of normal soft tissue. However, long-term volume retention is suboptimal (30-70%), often requiring multiple surgeries. [Read more…]
Stable expression in a cell lines is very useful for numerous projects. For example, it helps in Eukaryotes for optimization of protein productions and it is convenient for recurrent needs and high yields. Stable exogenous expression of tagged protein allows extensive tracking and localisation of the protein in live. Cell lines stably expressing a protein can be used for various screenings such as lost of function assays (CAS9-expressing cells), and reporter-based assays (Gaussian luciferase). Of course, the stable expression could also be shRNA for knock-down.
Stable expression implies insertion into the genome. It used to be random and could cause hits with side effects. Now, targeted insertion in Safe-Harbor sites is now possible. How can you take advantage of this progress?
Mouse ROSA26 and Human AAVS1 safe harbor sites
First of all, what is a safe-harbor site? It is an ideal site in the genome in which we can add a construct without harm and expect consistent level expression. For years, researchers have looked for it. In 1997, Zambrowicz et al initiated the discovery of the ROSA26 site on Mouse chromosome 6. It was shown it is a transcriptionally active region with an open chromatin configuration and transgene insertion has no or minimal effect on global and local gene expression. Remarkably, a ROSA26 inserted transgene is expressed in all tissues.
Similarly, in the human genome there is a safe-harbor site on chromosome 19 (locus PPP1R12C) called AAVS1. It was described more recently by DeKelver, et al. (2010). It has become a remarkable safe harbor site for ESC (Embryonic Stem Cells) and iPSC (induced Pluripotent Stem Cells) because of the robust expression of harboring constructs and the absence of abnormalities or differentiation deficits.
Since absence of visible effect doesn’t mean there is no at all risk of effect, I should mention that an ideal genomic safe-harbor doesn’t exist yet. Nevertheless, AAVS1 in human and ROSA26 are certainly the safer harbor sites today.
Comprehensive Safe-Harbor kit
Today we can easily insert a construction into a targeted site of the genome and so maintain its integrity. The principle is based on the targeted insertion of a Donor construction as illustrated in figure 1.
Each kit includes vectors expressing the system (CRISPR-CAS9) and the following:
- Donor vector in which you clone your ORF of interest
- RFP Donor control to monitor in fluorescence the knock-in
- Primer pairs for the PCR analysis of the genome integration
You can also obtain the Donor vector with your ORF of interest upon request.
The RFP control is highly convenient allowing a direct monitoring of the transgene genome integration (Figure 2).
Comparing with the control (without Donor), we can see the high integration efficiency after only 12 days of puromycin selection.
Comparing CRISPR-CAS9 Safe-Harbor to the classic method
Well… Think easier, faster, more reliable and cleaner!
So, is it a new way to work? As soon as you get the Safe-Harbor kit (with the empty Donor vector), you can use it indefinitely to establish promptly isogenic and polyclonal cell lines expressing all the ORFs you need. And furthermore the cell lines will be more reliable than random integration of lentivirus or plasmid.
Any questions? Please feel free to get in contact by leaving your comments below!
Induced-pluripotent stem cells (iPSC) are produced from a variety of source tissues including fibroblasts, epithelial progenitor cells, peripheral blood mononucleocytes (PBMC), and others. For Human iPSC, the pluripotency state is sometimes referred to as the “primed” state. Among iPSC lines, heterogeneity has been shown to exist in proliferation and the capacity to differentiate. This can cause issues in data interpretation or even limit the utility of Human iPSC in some disease cell models for basic research or drug discovery.
Recent publications have shown that Human iPSC, when cultured under special conditions, can be transitioned to what may be a more primitive form perhaps similar to cells in the pre-implantation state of a developing embryo. This state is referred to as the “naïve” state. In mouse and rabbit iPSC systems, naïve state cells have been shown to increase the efficiency and reproducibility (less bias) of terminal differentiation, including giving rise to some terminally differentiated cell types that display greater maturity. Thanks to this evidence (in other species), the accelerated growth characteristics of naïve cells, and other attributes linked to pluripotency, there is worldwide a rapidly growing interest in the Human stem cell research community. [Read more…]
The sensitivity and specificity of the primary and secondary antibodies used together with the IHC procedure used, are critical to avoid biased results. Several factors can cause false-positive or false-negative data, so they should all be verified as much as possible for each experimental set-up.
- Detection of the antigen of interest by the primary antibody
- Detection of the primary antibody by secondary antibodies
- Tissue preparation
Here, let’s look at 3 tips that will be of help to improve your IHC data. [Read more…]
The CRISPR-CAS9 system may well have opened Pandora’s box, but it is also definitely the cornucopia of genome editing.
We can do what we want in the genome: settle a mutation, correct a mutation, insert a fluorescent tag to a protein, add an exogenous gene, delete an endogenous function, suppress a cis-regulatory region, add a reporter…. I might just not have enough imagination!
The main challenge is to define a good strategy, taking in account the specifics of the project and being aware of the corresponding limitations. [Read more…]