Liver Cells from Circulating Blood Cells Under Clinically Safe Conditions


Can we convert circulating blood cells into working liver cells? Think of what this would mean for people who have liver problems. While is sounds like science fiction, the laboratory of James Ross at the University of Edinburgh, in collaboration with other scientists, has managed to do exactly that.

Ross and his colleagues developed an efficient method for converting circulating white blood cells into induced pluripotent stem cells (iPSCs). As previously mentioned on this blog, iPSCs are made from mature, adult cells by genetically engineering those cells with a cocktail of genes (in this case Oct4, Sox2, Klf4, L-Myc, and Lin28), and then culturing the cells in a special culture system that allows them to grow and become pluripotent stem cells that can theoretically differentiate into any of the 210 adult cell types in the human body.

Since the production of iPSCs from mature cells requires the insertion of particular genes into those cells, scientists typically use viruses or other vehicles to do this, which can introduce mutations into the genomes of the cells. Ross and his coworkers, however, used a non-integration method for reprogramming fresh or frozen white blood cells. They inserted small circles of DNA called “episomes” into these cells using a technique called “electroporation,” which binds the DNA to the surfaces of the cells and then subjects them to an electrical pulse that quickly moves the DNA into the cells without harming them. The genes on the episome are then expressed, but only transiently, which is all that is required to reprogram the adult cells into iPSCs. The cells were also cultured in a feeder-free system, which means that no animal products were involved in the production of these iPSC lines.  This constitutes, so-called “Good Manufacturing Practice” or GMP, which is required is a product is to be used for human patients.

Ross and others achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×105 seeded MNC), and when they used the same protocol to cord blood or fetal liver-derived blood-making (CD34+) cells, they achieved a reprogramming rate of 0.148% (148 iPSC colonies from 105 seeding cells). These iPSC lines were then used to make differentiated liver cells. This procedure tends to produce quasi-liver cells that do not have the characteristics of mature liver cells, but in this case, Ross and others derived cells that have proper drug metabolic function. This suggests that the iPSC-derived liver cells were at least mature enough to express many of the enzymes necessary to properly metabolize drugs. While these cells were probably not fully mature, they were a good deal further along than those derived in other experiments.

These experiments show that it is feasible to make liver cells for drug screen from circulating blood cells in a manner that is clinically safe. It is presently unclear if these cells can serve as material to heal a damaged liver, and that will take more work. Also, this procedure almost certainly would cost a good deal of money, and for that reason, banked iPSCs from white blood cells that have been fully tissue typed might be a better way to use cells made in this manner.

See Jing Liu, and others, Experimental Cell Research, 6 August 2015, Article ECR15383.

Umbilical Cord Stem Cells Preserve Heart Function After a Heart Attack in Mice


A consortium of Portuguese scientists have conducted an extensive examination of the effects of mesenchymal stromal cells from umbilical cord on the heart of mice that have suffered a massive heart attack. Even more remarkable is that these workers used a proprietary technique to harvest, process, and prepare the umbilical cord stem cells in the hopes that this technique would give rise to a commercial product that will be tested in human clinical trials,

Human umbilical cord tissue-derived Mesenchymal Stromal Cells (MSCs) were obtained by means of a proprietary technology that was developed by a biomedical company called ECBio. Their product,, UCX®, consists of clean, high-quality, umbilical cord stem cells that are collected under Good Manufacturing Practices. The use of Good Manufacturing Practice means that UCX is potentially a clinical-grade product. Thus, this paper represents a preclinical evaluation of UCX.

This experiments in this paper used standard methods to give mice heart attacks that were later received injections of UCX into their heart muscle. The same UCX cells were used in experiments with cultured cells to determine their effects under more controlled conditions.

The mice that received the UCX injections into their heart muscles after suffering from a large heart attack showed preservation of heart function. Also, measurements of the numbers of dead cells in the heart muscle of heart-sick mice that did and did not receive injections of umbilical cord cells into their hearts showed that the umbilical cord stem cells preserved heart muscle cells and prevented them from dying. Additionally, the implanted umbilical cord MSCs induced the growth and formation of many small blood vessels in the infarcted area of the heart. This prevented the heart from undergoing remodeling (enlargement), and preserved heart structure and function.

When subjected to a battery of tests on cultured cells, UCX activated cardiac stem cells, which are the resident stem cell population in the heart. Implanted UCX cells activated the proliferation of cardiac stem cells and their differentiation into heart muscle cells. There was no evidence that umbilical cord MSCs differentiated into heart muscle cells and engrafted into the heart. Rather UCX seems to help the heart by means of paracrine mechanisms, which simply means that they secrete healing molecules in the heart and help the heart heal itself.

In conclusion, Diana Santos Nascimento, the lead author of this work, and her colleagues state that, “the method of UCX® extraction and subsequent processing has been recently adapted to advanced therapy medicinal product (ATMP) standards, as defined by the guideline on the minimum quality data for certification of ATMP. Given that our work constitutes a proof-of-principle for the cardioprotective effects UCX® exert in the context of MI, a future clinical usage of this off-the-shelf cellular product can be envisaged.”

Preclinical trials with larger animals should come next, and after that, hopefully, the first human clinical trials will begin.

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.”