3D cellular culture

Several trials have been already done to produce a three dimensional support to grow cells. Collagen matrix, gelatin, nanofibers are some examples and each of them has some problems or limitations that have not allowed to be selected as a method of choice for three dimensional cell culture up to date. The advantage of three dimensional cell culture is to restore the normal environment where one cell is located into the tissue or in organism. The monolayer on a plastic surface or a suspension avoids the interaction with matrix or with other cells and, as a consequence, it affects cellular behavior. Furthermore, the plastic surface cannot be deformed in a similar way like the matrix and cells partially loss their plasticity. Matrigel and gelatin or collagen layer may help to maintain the three dimensional conformation of cells, but at the same time they are not totally inert surface and may activate some signaling pathways usually silenced. A possible alternative to matrigel or collagen layer is paper. Yes, the normal chromatographic paper has been proposed as a possible support to grow three dimensional cell culture. A recent paper published in PNAS journal describes this protocol. Scientists soaked the chromatography paper in hydrogel and cells and papers were layered at the bottom of tissue dish. In this way, cells were able to grow in contact with other cells cultured in the upper paper. To analyze one layer it was possible to peel the paper from the stack and work on it. The major advantage of the method, rather the low cost, is to sample a living tissue without fixing it or making thin sections. You can split the tissue and put inside the paper layer and when you want to analyze it, you just have to open it. In contrast, the main limitations are the lack of vascularization that is normally present in the natural tissue and the impairment of fluid flow. Thus, further work has to be done to make possible the local fluid flow that is really important in certain studies to study, for instance, the pharmacodynamic of drugs. Of course, even if this model is really interesting, as well as other three dimensional supports, it cannot substitute the use of animal model in science for many reasons. In particular, animals have vasculature, time dependent physiological and pathological remodeling, inflammation and immune system that are not present in any three dimensional in vitro model. All these elements are fundamental to understand the biological mechanism of pathways and characterize new drugs. In conclusion, paper support seems a very interesting model to study preclinical development of drug or to deep cell cell interactions and further applications will be published soon.

Embryonic stem cells in diabetes therapy

Beta cells transplantation seems the most promising therapy for diabetes, because other current treatments loss their favorable advantage and can not avoid serious adverse effects, such as hyperglycemia. Embryonic stem cells are the ideal biological tool to achieve the complete restoring of pancreatic islet. Current research on diabetes is focused on the identification of a protocol to culture cells. Indeed, embryonic stem cells must be differentiated in order to produce insulin. It has been recently published that high concentration of exendin 4 are able to induce insulin synthesis and differentiation to pancreatic cells. This strategy is a modification of the original protocol that requires the simultaneous administration of exedin 4, nicotinamide and activin b. Additional culturing in low glucose medium seems to further improve embryonic cells differentiation. Despite previous studies, Hu and coworkers (Acta Pharmacologica Sinica) obtained more insulin 1 and peptide C. This study gives great hope to patients that suffer for diabetes and can not have a benefit from traditional approaches. Embryonic stem cells must be further studied to improve culturing conditions in order to achieve cellular population useful for transplantation.

Nuocytes, a novel cell type

A recent work published in Nature demonstrates the existence of a new class of cells, namely noucytes. This cells seem really important for the type II immuninity. The type II immunity protects our body from parasitic helminth infections, by promoting the release of type II cytokines, activating eosinophiles and goblet cells and secreting mucus and IgE.
The same system is also responsible for asthma and other allergic disfunctions. Nuocytes are a novel component of innate immune system and seem involved in IL13 release during the first steps of helminth infection. Nuocytes primarily respond to IL25 and IL33 stimuli either in vitro or in vivo, then start the immune response. Lack of IL25 ad IL33 cytokines determines nuocytes expansion failure and lastly severe defect in worm expulsion. Thus, nuocytes play a crucial role in body defense and this and subsequent studies will help us to understand better the mechanism of action of innate immune system. Nuocytes will represent a new possible target for immune stimulating molecules in order to increase the protection versus infection, as well as it could be possible that new anti asthma drugs will be directly act on this class of cells.

Hela cells, a story lasting 60 years.

Henrietta Lacks died in 1951 at the Johns Hopkins hospital in Baltimore because of an aggressive form of cervical cancer. She would be probably unknown now, if your cells hadn’t been extracted and cultured as HeLa cells. Scientists of every molecular biology and cellular lab know about these cells because they have used them at least one time or because they have studied their application on biology books. However, it’s very interesting the history about HeLa cells. Henrietta was 31 years old when she died and she had five children. She was the unwitting protagonist of the story, because on 1950s none informed consent was asked her. Doctor Gey and his wife had all scientific merits to make possible HeLa cell culture. They put these cells on Petri dishes; at that time they were performing a lot of experiments to try to culture human cell lines. HeLa cells were able to quickly grow in established conditions, differentially from other cells tested. Dr Gey sent his cells to many laboratories around the world and shared information about culture conditions and so on. This generosity allowed important scientific advances, especially in vaccination field because HeLa cells were firstly used to test and produce the Polio vaccine. Unfortunately, giving their ability to grow also in unfavorable conditions, HeLa cells became one of the most dangerous contaminant agents of other cell lines. The doubt that scientists were using HeLa cells in their experiments rather than breast, prostate or placental cells made necessary further analyses to figure out the true identity of cell lines used. Thus, after almost thirty years from her death, the Hopkins Hospital contacted Henrietta’s children and familiar to invite them donate some blood or tissue samples.
Genetic analyses and blood type were information required by scientists to complete the Henrietta’ profile and recognize HeLa cells from others. Even if the scientific purpose was correct, Henrietta’ family didn’t have all explanation needed to well understand physicians’ operations. This was only one of dark points from an ethical point of view present in this story. Fortunately after Henrietta’s experience, ethical question has acquired great importance in experimental medicine and now informed consent is required for every medical action. Another important issue of this story were the moral and legal questions that arose about the commercial value of something derived from human body. Who may have the copyright of HeLa cells? With these cells several billions of dollars have been gained by pharmaceutical companies, research institutes and so on, but Henrietta’s family haven’t had any benefit. But on other hand, what has been the role of Henrietta in whole story? She was just a poor mother who died too soon.

CXCR1 in breast cancer

Scientists from the University of Michigan and the INSERM (France) identified Cxcr1 gene as differentially expressed in cancer stem cells in comparison with other cancer cells in breast malignancies.
CXCR1 is the receptor for interleukin 8 and has been involved in tumour progression and metastasis in several kinds of cancers, such as prostate, glioma, ovarian and breast cancers. Furthermore, IL8 has been implicated in self renewal of stem cells in vitro. French researchers tested some small molecule inhibitors targeting CXCR1 or some antibody against this receptor in order to evaluate the effect on cellular behavior. These inhibitors directly acted versus cancer stem cells and at the same time indirectly induced cell death in bulk tumour. The promising result was that the whole cancer population was eliminated. A possible explanation for this larger effect could be the release of FAS ligand after inhibiting CXCR1 that determined apoptosis in all cancer cells. In animal model CXCR1 inhibitors reduced tumour mass and blocked metastasis, either alone or in combination with other drugs. Another possible approach to block cancer progression by acting on CXCR1 pathway is to interfere with IL8 production. Scientists from the University of Texas demonstrated that siRNA-IL8 reduced tumour weight in comparison to control in animal models. These studies seem really promising but further proof is necessary to validate CXCR1 pathway as a target for therapeutic intervention.

Novel function for IHD1 in glioma

Genetic mutations are usually identified in cancer. These alterations can cause loss of function, when one mutation interferes with protein functionality, or gain of function when a mutated protein over-works or is over-expressed in a wrong cellular district. In this case, a protein maintains the same function as a normal one. In some cases, mutation can generate novel functions in a protein.
This is the case of IDH1, the isocitrate dehydrogenase 1 that usually converts the isocitrate into alpha- ketoglutarate. When this protein is mutated, an overproduction of 2 hydroxyglutarate (2HG) is abundantly recovered into cells. 2HG is toxic for brain and its presence is correlated with cancer. X-ray diffraction demonstrated that the mutation at arginine 132 results in the formation of a distinct active site, different from those that catalyzes the normal reaction. So, a novel mutation has been identified and a novel function has been recognized. IDH1 could be considered a new target for therapeutic intervention. A selective and specific small molecule inhibitor could interact only with the mutated form, without interfering with the wild type protein and the glucose metabolism. This discover could hopefully help scientists to identify a treatment for glioma.

One odd and dramatic case in medicine

In 2007 a young Japanese woman gave birth to her child and after only one month died for leukaemia, ABL-BRC positive. During her pregnancy she didn’t know to be sick and also doctors didn’t diagnose the disease. After eleven months, also the baby showed a tumour into his cheek. Physicians excluded a possible sarcoma and diagnosed also in this case leukaemia, ABL-BCR positive, the same mother’s disease. Further analyses demonstrated that no genetic material from the father was present in baby’s tumour, this means that cancer cells derived only form the mother and escaped the foetal barrier into uterus. Scientist has suspected that some forms of cancers, such as leukaemia and melanoma that are prompt to metastasize, could move form mother to fetus and this unfortunate story gave the genetic proof. Cancer cells can escape our immune system and also foetal barrier, knowing molecular basis of this mechanism could help us to better understand cancer biology and find new strategies for therapeutic intervention. Fortunately, there are few possibilities that the case of Japanese woman can repeat and we can learn a lot from odd examples like this. A positive note to conclude this post: the baby was successfully cared and is still alive.

Comparison between Baculovirus and E. Coli, as expression system

Baculovirus expression system is commonly used for protein expression, in particular it is used for eukaryotic proteins that cannot be correctly folded in E. Coli. This virus, not infectious for human, infect insect cells, such as Sf9, Sf21 or Hi5, and are isolated to performed high amount of recombinant proteins. Indeed, Baculovirus genome could be easily modified with current molecular biology techniques –molecular cloning, following by recombination and transposition- and propagated in normal cellular biology lab, not P2 or P3 chambers are necessary to use this virus. It’s possible to insert long fragment of exogenous DNA into virus genome, this aspect is important and represent a good advantages in respect of E. Coli because eukaryotic proteins are usually codified by long genes.

Another significant advantage is the capability to introduce post-translational modifications, such as phosphorylation, glycosilation that are necessary to obtain the complete functionality of the recombinant protein. Thus, proteins produced with this system could be employed for instance in biochemical assays, after purification, and in screening of pharmacological compounds. By contrast, the main disadvantages associated with Baculovirus are the cost and the yield: insect cells culture is more expensive than E. Coli culture, because of the media, sera and molecular reagents necessary to make the transfection. Furthermore, the yield is lower and if 1 mg of recombinant protein can be produced form 1 litre of E. Coli culture, much more litres of insect cells are necessary to produce the same amount of protein.
What are the parameters important to choose one type of expression system in respect of another one? Firstly, it has to be known very well the protein that has to be produced: literature review gives interesting suggestions and the most common expression system is usually the best one. Secondarily, the aim of the study is important, indeed if a functional study has to be performed, it’s crucial to have an active protein also in a little amount, by contrast for crystallization study is important to consider that milligrams of protein have to be purified. Finally, both Baculovirus and E. Coli are tool easy to use, but in the case of Baculovirus some experience in cellular biology rather than biochemistry is important. The timing has also to be considered because Baculovirus-insect cell system requires at least one month, after cloning, to perform first infections; by contrast, E. Coli needs only cloning procedures to express the protein of interest. Both recombinant viruses and bacteria can be frozen, in order to have reagents ready to use, it’s important keep sterility and a great classification if numerous proteins are studied at the same time in the lab: special software are commercially available for this purpose. In conclusion, both techniques are useful in biochemistry, structural biology and molecular biology and in respect of the aim of the study is crucial to choose the more convenient one.

Minicells to target tumour cells

A difficult challenge in cancer therapeutics is to develop drugs that can overcome the heterogeneity and resistance of cancer cells. A tumor usually consist on several type of cells that contribute to tumor growth, tissue invasion and metastasis in different way. Furthermore, during a treatment drugs become ineffective because of survival of cells that escape death induced by drug.

Small minicells derived form bacteria was used to targeted delivery of drug into cancerous cells. Antibodies can target minicells to tumor cell surface receptors and release drug at the specific site. A recent publication demonstrated how it was possible to subsequently administrate short hairpin RNA and cytotoxic drug in order to firstly knock down a multidrug resistance protein, then kill the cells made vulnerable. This strategy was tested on xenograft tumor in mice model and gave the opportunity to tune the dosage of cytotoxic drug, diminishing adverse effects. Minicells were not toxic for animals and didn’t compromise their survival: this approach seems promising to specifically treat resistant tumor and the use of shRNA allows to personalize the treatment. Other important challenges in next future will be the discovery of other route to target minicells, because resistance mechanism will be early developed to decrease receptor expression on cell surface.

Challenges in pluripotency

Pluripotency is a special feature of stem cells to renew and generate part of the body. Totipotency is characteristic of embryonic stem cells that can generate each organ or tissue, while pluripotency is more limited and only few tissue can be product by stem cells. Four factors seem to control pluripotency: Oct4, Klf4, Myc, Sox2.

All these genes are crucial during embryonal development and in some cases mutations could lead to serious diseases, such as cancer for Myc gene, or impaired development for Oct4 and Sox2. A recent work published on Nature journal demonstrated that, by using these four factors (previously used to reprogram fibroblast), it could be possible to generate human pluripotent stem cells from fetal, neonatal and adult primary cells. Human induced pluripotent cells showed similar features of embryonic stem cells in terms of morphology and gene expression and were able to form teratomas when injected in immuno-deficient mice. This work opens new perspectives in stem cells research: for instance in next future, it could be possible to culture primary cells directly form patients and induce pluripotent cells in order to repair tissue, make transplantation or care degenerative diseases without troubles of rejection.