Celprogen Markets a Xeno-Free Medium for Stem Cells


Celprogen Inc., has designed a cell culture medium for growing stem cells of all types in the laboratory that does not contain any animal products. Such a medium is called “Xeno-free.”

This new xeno-free medium, XFS2 can successfully culture induced pluripotent stem cells, primary human cells, stem cells and progenitor cells. XFS2 can also be used to grow cancer cell for research purposes.

Growing stem cells in cell culture requires a unique mixture of growth factors that stimulate cell proliferation and cell survival. Because stem cells can grow in XFS2 without differentiating, it can be used to grow cells for clinical trials s well.

Celprogen scientists have used XFS2 to grow human embryonic stem cells, fat-derived stem cells, and stem cells isolated from specific human organs. A chief advantage of XFS2 is that it does not contain any human serum, which is essential for clinical applications, since patients could have serious immunological reactions against serum that does not come from their own blood.

When stem cells grown in XFS2 were compared with stem cells grown in other cell culture media, the XFS2-grown cells did not display any detectable differences from their serum-grown counterparts. Furthermore, according to Celprogen, stem and progenitor cells grew robustly in XFS2. We will certainly need to see the reactions of laboratories who chose to use XFS2 to grow their stem cells before we can confirm or deny this.

Celprogen hopes that their safe and xeno-free XFS2 medium will facilitate the potential application of stem cell transplantation for the treatment of various diseases, and Celprogen is equally hopeful that their new medium will prove itself to be safe, give reproducible results, and a high-quality medium for the propagation of stem and progenitor cells in the laboratory.

Has anyone used XFS2 to grow their stem or progenitor cells in the laboratory yet?  If so, let me know how it works.

Safer Culture Conditions for Stem Cells


Jeanne Loring from the Scripps Institute is the senior author of a very important study that examined the culture conditions for pluripotent stem cells.

Several scientists have discovered that induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) can accumulate cancer-causing mutations when grown in culture for extended periods of time (for example, see Uri Weissbein, Nissim Benvenisty, and Uri Ben-David, J Cell Biol. 2014 Jan 20; 204(2): 153–163). However, some laboratories have managed to keep ESCs in culture for extended periods without observing instabilities.

To try to tease apart why this might be the case, Loring and her group examined various culture methods and determined that some stem cell culture methods are associated with increased incidence of mutations in the DNA of stem cells.

“This is about quality control; we’re making sure these cells are safe and effective,” said Loring, who is a professor of developmental neurobiology at Scripps Research Institute (SRI) in San Diego, CA.

All cells run the risk of accumulating mutations when they divide, but previous research from Loring and her colleagues showed that particular culture conditions could potentially select for faster growth and mutations that accelerate growth. Such growth-enhancing mutations are sometimes associated with tumors.

“Most changes will not compromise the safety of the cells for therapy, but we need to monitor the cultures so that we know what sorts of changes take place,” said Ibon Garitaonandia, who is a postdoctoral research fellow in Loring’s laboratory at SRI.

New research from Loring’s group has shown how particular culture conditions can reduce the likelihood of mutations. Loring and her colleagues tested several different types of surfaces upon which the cells were grown. They also used different ways of propagating or “passaging” the cultures. When cells are grown in culture, the culture dishes must be scraped to get the cells off them and then the cells must be transferred to a fresh culture dish. How you do this matters: do you use enzymes to detach the cells, or do you mechanically scrape them off? Other culture techniques use layers of “feeder cells” that do not divide, but are still able to secrete growth factors that improve the health of the growing stem cells.

Loring and her crew tested various combinations of surfaces, passaging methods and feeder cell populations and grew the cells for three years with over 100 passages. Over the course of this experiment, the cells were sampled and analyzed for the presence of new mutations in their genomes.

It turns out that stem cells grown on feeder cells that are passaged by hand (manually) show the fewest growth-enhancing mutations after being cultured for three years.

Loring’s study also demonstrated the importance of monitoring cell lines over time. In particular, deletion of the TP53 gene, a tumor suppressor gene, in whose absence cancer develops, should be closely watched.

“If you want to preserve the integrity of the genome, then grow your cells under those conditions with feeder cells and manual passaging,” said Loring. “Also, analyze your cells. It’s really easy, she added.

When Thomson made the first human ESC lines, he used feeder cells derived from mouse skin cells.  However, the use of animal materials to make ESCs might pollute them with animal viruses and specific sugars from the surfaces of the animal cells might also contaminate the surfaces of the ESCs, making them unsuitable for regenerative medicine (see Stem Cells 2006; 24:221-229).  To address this problem, several laboratories have made “Xeno-free” ESC lines that were made without touching any animal products.  Some of these Xeno-free lines were made without feeder cells (see C. Ellerström, et al., Stem Cells. 2006 Oct;24(10):2170-6)., but others were made with human feeder cell lines (see K Rajala, et al., Hum Reprod. 2007 May;22(5):1231-8). Therefore, it appears, that the use of human feeder cell lines are preferable to feeder-free systems, given Loring’s findings.  However, it is also possible that such culture systems are also preferable for iPSCs, which do not have the problem of immunological rejection for patients, and do not require the killing of the youngest members of humanity.  Therefore, Loring’s work could very well benefit iPSC cultures as well.

Skin Tissue Grown From Human Stem Cells


A research team from King’s College, London, in collaboration with the San Francisco Veteran Affairs Medical Center has succeeded in growing the epidermal layer of skin in culture, this cultured skin has many of the mechanical and biological properties of actual human skin.

The outermost layer of the skin, known as the epidermis forms a protective barrier between the external environment and the body. It protects against water loss and prevents the entry of microorganisms.

Tissue engineers have been able to grow skin cells (keratinocytes) in culture, but getting them to organize into an organ that resembles biological skin has proven rather difficult. However, the ability to test drugs on cultured skin that greatly mimics human skin has been the goal of such research for several years.

For this present project, keratinocytes were made from induced pluripotent stem cells that were derived from skin cells obtained from biopsies. These keratinocytes made from induced pluripotent stem cells (iPSCs) were very similar to keratinocytes made from embryonic stem cells and primary keratinocytes isolated from skin biopsies.

To form a three-dimensional structure like skin, the keratinocytes were cultured in a high-to-low humidity environment and they assembled into a layer structure that looked like human skin. When this cultured skin was compared with skin made from embryonic stem cell-derived keratinocytes or from keratinocytes isolated from skin biopsies, there were no significant structural differences.

Scientists hope to use this cultured skin to study congenital skin diseases like ichthyosis (characterized by dry, flaky skin) or atopic dermatitis. Growing large quantities of skin in culture will also allow drugs and cosmetics to be effectively tested for safety without the use of expensive and sometimes highly variable animal models.

This technology would also allow different laboratories to grow skin from different ethnic groups that have distinct types of skin with variable biological properties.

Induced Pluripotent Stem Cells Replace Liver Function in Mice


Liver transplants save lives and in the United States there is a shortage of livers for transplantation. Between July 1, 2008 and June 30, 2011, well over 14,601 adult donor livers were recovered and transplanted. Of these livers that were transplanted, many other patients died from liver failure. If there was a way to restore liver function in patients with liver failure without dependence on a liver from a liver donor, then we might be able to extend their lives.

A paper from the laboratory of Hossein Baharvand at the University of Science and Culture in Tehran, Iran provides a step towards doing just that. In this paper, Baharvand and his colleagues used human induced pluripotent stem cells to make hepatocyte-like cells or HLCs. Hepatocyte is a fancy word for a liver cell. These HLCs were then transplanted into the spleen of mice that have damaged livers, and they rescued liver function in these mice.

The liver is a vital organ. It processes molecules absorbed by the digestive system, processes foreign chemicals to make them more easily excreted. It also produces bile, which helps dispose of fat-soluble waste and solubilize fats for degradation in the small intestine during digestion. It also produces blood plasma proteins, cholesterol and special proteins to cholesterol and fat transport, converts excess glucose into glycogen for storage, regulates blood levels of amino acids (the building blocks of proteins), processes used hemoglobin to recycle its iron content, converts poisonous ammonia to urea, regulates blood clotting, and helps the body resist infections by producing immune factors and removing bacteria from the bloodstream. Thus without a functioning liver, you are in deep weeds.

Induced pluripotent stem cells or iPSCs are made from adult cells that have been genetically engineered to de-differentiate into embryonic-like stem cells. They can be grown in culture to large numbers, and can also be differentiated into, potentially, any cell type in the adult body.

In this paper, Baharvand and his colleagues grew human iPSCs in “matrigel,” and then grew them in suspension. Matrigel is gooey and the cells stick to it and grow, and they were grown in matrigel culture for 1 week. After one week, the cells were grown in liquid suspension for 1-2 weeks. The cells have better access to soluble growth factors in liquid culture and tend to grow faster. After this they were grown in a stirred culture (known as a spinner).  This expanded the cells into large numbers for further use.

 Expansion and characterization of human induced pluripotent stem cells (hiPSCs) in a dynamic suspension culture. (A) Schematic representation of the suspension culture and expansion of human pluripotent stem cells (hPSCs) from adherent to stirred bioreactor. The cells were transferred to bacterial dishes to adapt to three-dimensional (3D) environment and then transferred into the dynamic phase, stirred flask. (B) Morphology of a tested hiPSC line (hiPSC1) after passage in a stirred suspension bioreactor as monitored by dark-field microscopy. (C) Immunostaining of cross sections of spheroids for OCT4 and TRA-1-81. Scale bar: 50 μm. (D) Flow cytometry analysis and (E) normal karyotype of suspended cells in the bioreactor.
Expansion and characterization of human induced pluripotent stem cells (hiPSCs) in a dynamic suspension culture. (A) Schematic representation of the suspension culture and expansion of human pluripotent stem cells (hPSCs) from adherent to stirred bioreactor. The cells were transferred to bacterial dishes to adapt to three-dimensional (3D) environment and then transferred into the dynamic phase, stirred flask. (B) Morphology of a tested hiPSC line (hiPSC1) after passage in a stirred suspension bioreactor as monitored by dark-field microscopy. (C) Immunostaining of cross sections of spheroids for OCT4 and TRA-1-81. Scale bar: 50 μm. (D) Flow cytometry analysis and (E) normal karyotype of suspended cells in the bioreactor.

Getting cells to grow in liquid suspension tends to be a bit of an art form, but these iPSCs grew rather well. Also, the iPSCs were differentiated into definitive endoderm, which is the first step in bringing cells to the liver cell stage. The drug Rapamycin and activin (50 ng / L for those who are interested) were used to bring the growing iPSCs to the definitive endoderm.  The cells expressed all kinds of endoderm-specific genes.  Endoderm is the embryonic germ layer from which the digestive system and its accessory organs forms.

 Induction of hiPSCs into definitive endoderm. (A) Diagrammatic representation of the experimental groups for endoderm induction of hiPSCs in the bacterial dish static suspension, which include rapamycin (Rapa) “priming” and activin A “inducing” phases, and positive control groups of hiPSCs cultured in the absence of Rapa in suspension or adherent cultures in the presence of activin A. (B) Gene expression analysis of hiPSCs induced into endodermal cells. Quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) showed no significant differences in SOX17 and FOXA2 [definitive endoderm (DE) markers], and BRA (mesoendoderm marker) in all groups. SOX7 (visceral endoderm marker) was expressed at a low level. Influence of the refreshment strategy of the induction medium on DE formation in suspension cultures by qRT-PCR (C), immunostaining (D), and flow cytometry (E). We compared single (SR), double (DR), and triple (TR) refreshment of induction medium per 24 h for 4 days after Rapa administration in the static suspension of hiPSCs. Scale bar: 100 μm. The target gene expression level in qRT-PCR was normalized to GAPDH and calibrated with (presented relative to) hiPSCs. Data are presented as mean±SD. Statistical analysis as determined by one-way ANOVA with Tukey's post hoc test, n=3 for B, C, and E. *P<0.05, **P<0.01.
Induction of hiPSCs into definitive endoderm. (A) Diagrammatic representation of the experimental groups for endoderm induction of hiPSCs in the bacterial dish static suspension, which include rapamycin (Rapa) “priming” and activin A “inducing” phases, and positive control groups of hiPSCs cultured in the absence of Rapa in suspension or adherent cultures in the presence of activin A. (B) Gene expression analysis of hiPSCs induced into endodermal cells. Quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) showed no significant differences in SOX17 and FOXA2 [definitive endoderm (DE) markers], and BRA (mesoendoderm marker) in all groups. SOX7 (visceral endoderm marker) was expressed at a low level. Influence of the refreshment strategy of the induction medium on DE formation in suspension cultures by qRT-PCR (C), immunostaining (D), and flow cytometry (E). We compared single (SR), double (DR), and triple (TR) refreshment of induction medium per 24 h for 4 days after Rapa administration in the static suspension of hiPSCs. Scale bar: 100 μm. The target gene expression level in qRT-PCR was normalized to GAPDH and calibrated with (presented relative to) hiPSCs. Data are presented as mean±SD. Statistical analysis as determined by one-way ANOVA with Tukey’s post hoc test, n=3 for B, C, and E. *P<0.05, **P<0.01.
After the cells went through this culture protocol, they were grown in a stirred liquid culture called a “spinner.” The culture system contain a cocktail of growth factors that differentiated the definitive endoderm cells into HLCs.  The cells formed little spheres that expressed a host of liver-specific genes.

Differentiation of hepatocyte-like cells (HLCs) from hiPSCs in the stirred bioreactor. (A) Stepwise protocol for differentiation of hiPSCs into HLCs. (B) The morphology and cross section of spheroids at day 21. The spheroids in this step were observed as cystic and dense spheroids. Hematoxylin and eosin (H&E) staining of spheroid cross sections indicated cystic and dense epithelioid appearances in the transplant and dense spheroids, respectively. Scale bar: 100 μm. (C) Comparative relative mRNA expression in cystic and dense spheroids normalized to GAPDH and calibrated to undifferentiated hiPSCs. Transplant and dense spheroids expressed more early and late hepatic lineage markers, respectively. Data are presented as mean. n=3. (D) Transmission electron microscopy (TEM) of differentiated cells in dense spheroids at day 21. Nucleus (N), nucleoli (n), mitochondria (M), Golgi apparatus (G), lysosomes (Ly), rough endoplasmic reticuli (arrowhead), glycogen granules (GR), intermediate filaments (CK), tight junctions (TJ), gap junctions (GJ), fascia adherens (FA), junctional complex (JC), microvilli (MV), and bile-like canaliculus (BLC). Scale bar: 1 μm.
Differentiation of hepatocyte-like cells (HLCs) from hiPSCs in the stirred bioreactor. (A) Stepwise protocol for differentiation of hiPSCs into HLCs. (B) The morphology and cross section of spheroids at day 21. The spheroids in this step were observed as cystic and dense spheroids. Hematoxylin and eosin (H&E) staining of spheroid cross sections indicated cystic and dense epithelioid appearances in the transplant and dense spheroids, respectively. Scale bar: 100 μm. (C) Comparative relative mRNA expression in cystic and dense spheroids normalized to GAPDH and calibrated to undifferentiated hiPSCs. Transplant and dense spheroids expressed more early and late hepatic lineage markers, respectively. Data are presented as mean. n=3. (D) Transmission electron microscopy (TEM) of differentiated cells in dense spheroids at day 21. Nucleus (N), nucleoli (n), mitochondria (M), Golgi apparatus (G), lysosomes (Ly), rough endoplasmic reticuli (arrowhead), glycogen granules (GR), intermediate filaments (CK), tight junctions (TJ), gap junctions (GJ), fascia adherens (FA), junctional complex (JC), microvilli (MV), and bile-like canaliculus (BLC). Scale bar: 1 μm.

From the figure above, we can see that these HLCs, not only express liver-specific genes, but when they are examined in the electron microscope they look, for all intents and purposes, like liver cells.  Functional tests of these spheres of HLCs showed that they 1) took up low-density lipoprotein; 2) produced albumin (a major blood plasma protein); 3) expressed cytochrome P450s, which are the major enzymes used to process drugs; 4) produced urea from amino acids, just like real liver cells; 5) accumulated glycogen; 6) and made liver proteins (HNF4a, ALB, etc).

So it looks like liver, quacks like liver, but can it replace liver?  These HLCs were transplanted into the spleen of mice whose livers had been treated with carbon tetrachloride.  Carbon tetrachloride tends to make mincemeat of the liver, and these mice are in trouble, since their livers are toast.  Transplantation of the iPSC-derived HLCs into the spleens of these mice increased their survival rate and decreased the blood levels of liver enzymes that are usually present when there is liver damage.

This paper is significant because the procedure used provides an example of a “scalable” protocol for making large quantities of iPSCs, and their mass differentiation into definitive endoderm and then liver cells,  Because this can potentially provide enough cells to replace a nonfunctional liver, it represents a major step forward in regenerative medicine.

 

Plastic Bags Coated With Plasma For Culturing Stem Cells


Clinical products that are used to treat patients must be manufactured under a set of standards known as “Good Manufacturing Practice” or GMP. Drugs, catheters, stents, implants, pacemakers and so on must all be manufactured in a facility that strictly adheres to GMP standards and produces products that are consistently safe for patient use. Products made to GMP standards are free of contaminating microorganisms, free of molecules that cause robust rejection by the immune system, and known to be safe for use in a human patient.

Producing stem cells for regenerative medicine represent a tough case for several reasons. Most of the laboratory products sold off the shelf for tissue culture have some animal products in them, which disqualifies them for clinical use, since culture media with animal products can contain animal viruses or animal antigens that will cause patient’s immune systems to reject them. Growing stem cells under animal-free conditions is tedious, expensive, and the results are not always consistently reproducible. While some laboratories have made remarkable strides in growing cells under animal-free conditions, doing so in a manner that meets GMP standards is even more exacting.

New work by Kristina Lachman and Michael Thomas and their colleagues from the Fraunhofer Institute for Surface Engineering and Thin Films in Braunschweig, Germany, has shown that plastic bags coated with a plasma can provide excellent vessels for stem cell culture and can be manufactured in a manner that meets GMP standards.

The term “plasma” in physics refers to the state of a gas when a strong enough electric current is passed through it so that the gas mainly consists of ions. In such a state, the gas is no longer a gas and has properties unlike a solid, liquid or gas, but is considered a distinct state of matter. When a plasma is used to coat the inside surface of a plastic bag, it modifies the surface of the internal surface of the bag so that different types of cells can grow on it. The plasma also acts as a disinfectant while it transforms the surface of the bag so that cells are able to grow on it and even want to grow on it.

plasma_image

“Our goal was to realize a closed system in which cells grow undisturbed and without the risk of contamination. Coating the bags with plasma enables us to use them as a GMP laboratory,” said Henk Garritsen from Braunschweig Municipal Hospital.

To date, stem cells cultured from the patient’s own body have been grown in plastic culture dishes, spinner bottles, and bioreactors. However, systems like these, though initially sterile, must be opened in order to refill the culture medium or extract the cells. Every time the culture is opened there is a risk of contamination and the cells are rendered unusable.

Enter Werner Lindenmaier and Kurt Dittmar from the Helmholtz Center for Infection and Research who were already working on bags for stem cell cultivation. By collaborating with the experts at Fraunhofer who knew how to coat plastics with plasma, these scientists embarked on a very fruitful venture that culminated in experiments that showed that stem cells could robustly grow on plasma-coated films. Then a joint venture sponsored by the German Federal Ministry of Economics and Technology investigated the feasibility of plastic bags coated with plasma as a closed system for stem cells cultivation and growth.

To coat the bags with plasma, they are first filled with a non-reactive gas and then hit with low-voltage electrical currents. This generates a plasma in the bag and this plasma is a “luminous, ionized gas that chemically alters and at the same time disinfects the surface of the plastic,” said Lachmann.

The bags were coated in a pilot plant at Fraunhofer IST and then tested at the Helmholtz Center and the Braunschweig Municipal Hospital to test the diverse types of coatings used on the bags for their ability to support the growth of stem cells. Dittmat noted that, “We work with stem cells for bones, cartilage, fat, and nerves – the coating can be optimized for each of these cell types.”

The pilot plant at Fraunhofer IST has designed an automated system for making these stem cell culture bags. This automated system can make bags that are wholly reproducible in their composition and properties.

“We use medically approved bags for the coating,” said Thomas. “Nevertheless, the plasma treatment must be demonstrated to be innocuous before being approved for clinical use.”

The Role of Astrocytes in Lou Gehring’s Disease


A study from Columbia University and Harvard University has uncovered a complex interplay between neurons and support cells known as astrocytes that contributes to the pathology of ALS. Such an intricate interplay complicates regenerative therapies for this disease.

In the spinal cord, a group of neurons called motor neurons extend their axons to skeletal muscles and provide the neural signals for the muscles to contract, which allows movement. Motor neurons also have associated support cells known as glial cells, and a specific group of glial cells known as astrocytes associate with motor neurons in the spinal cord.

Astrocytes are star-shaped cells that surround neurons in the brain and spinal cord, and they outnumber neurons 50:1. Astrocytes are very active in the central nervous system, and serve to maintain, support, and repair the nervous tissue that they serve, and are responsible, in large part, for the plasticity of the nervous system.

astrocytes1 (1)

Motor neurons die off during the course of ALS, which leads to paralysis and death within two to fives years of diagnosis. ALS also affects neurons in the brain and it completely robs the individual of the ability to initiate movement or even breathe. There is, at present, no cure and no life-prolonging treatment for ALS.

Data from the ALS Association group suggests that astrocytes in ALS patients go from supporting neurons to strangling them. According to Lucie Bruijn, the chief scientist at the ALS Association in Washington D.C.,, these results seem to “strengthen the case that astrocytes are central to the ALS disease process.” She continued: “Furthermore, the results are based on an exciting new disease model system, one that will allow us to test important hypotheses and search for new therapeutic targets.”

In a cell culture system of ALS, in which neurons derived from embryonic cells were co-cultured with normal and ALS astrocytes, Bruijn’s team found that gene expression patterns in those neurons associated with ALS astrocytes were abnormal. In this experiment, neurons derived from embryonic stem cells with co-cultured with normal and ALS affected astrocytes. In a time course experiment in which gene expression profiles were analyzed from the neurons after specific amounts of time, the gene expression patterns from the normal astrocytes co-cultured with neurons were compared with those of the ALS-affected astrocytes co-cultured with neurons. From these experiments, it became clear that the ALS-affected astrocytes did not communicate properly with the nearby neurons.

Even though neurons communicated with each other by means of the release of neurotransmitters, astrocytes and other glial cells also communicate with each other by means of the release of various molecules. This astrocyte-neuron communication maintains healthy neuron function. However, in the case of ALS, the neuron-astrocyte communication is “profoundly disrupted” and is disruption is not neuron dependent, since in this experiment, the neurons were normal. Without proper communication with their astrocytes, motor neurons the spinal cords of ALS patients are not able to function properly.

According to Bruijn, “This study points out several potential points for treatment intervention.” The protection of motor neurons is the goal, since the astrocytes seem to be doing little to protect and support the neurons and also might be hurting them.

An added bonus to this study is that when spinal cords from mice with a disease that shows some similarities to ALS have their gene expression profiles compared to these gene expression profiles observed in the cultured neurons, the results are remarkably similar. This shows that culture system does recapitulate what goes on in the spinal cord.

The next step is to show that the molecular abnormalities discovered in this system mimics those that occur in human disease. This publication utilized mouse cells, and the human disease, while similar, is not exactly the same.