A More Efficient Way to Make Induced Pluripotent Stam cells

Mark Stadtfeld and his colleagues at the NYU Longone Medical Center has devised a new method for making induced pluripotent stem cells that greatly increases efficiency at which these cells are made.

Induced pluripotent stem cells or iPSCs are made from mature, adult cells by mean of a combination of genetic engineering and cell culture techniques. In short, the expression of four genes is forced in adult cells; Oct4, Sox2, Klf4, and c-Myc or OSKM. The proteins encoded by these four genes cooperatively work to drive a fraction of the cells into an immature state that resembles that of embryonic stem cells. These cells are them grown in cell culture systems that select for those cells that can grow continuously and form colonies of cells derived from progenitor cells. These cell colonies are them repeated isolated a re-cultured until an iPSC line has been established.

Unfortunately, this process is rather inefficient and tedious, since less than one percent or so of the reprogrammed cells actually undergo successful reprogramming. Additionally, it can take several weeks to properly establish an iPSC line. Thus, stem cell scientists have been looking at several different ways to boost the efficiency of this process.

Stadtfeld and his coworkers tried to add compounds to the cultured cells to determine if the culture conditions could actually augment the efficiency of the reprogramming process. “We especially wanted to know if these compounds could be combined to obtain stem cells at high-efficiency,” said Stadtfeld.

The compounds to which Stadtfeld was referring were two cell signaling proteins called Wnt and TFG-beta. Both of these compounds regulate a host of cell growth processes. Stadtfeld wanted to try regulating both of these pathways at the same time, in addition to providing cells with ascorbic acid, which is also known as vitamin C. Even vitamin C is more popularly known as an antioxidant, vitamin C also can remodel chromatin (that tight structure into which cells package their DNA).

When mouse skin fibroblasts were treated with OSKM and a compound that activates Wnt signaling, the efficiency of iPSC derivation increased slightly. The same thing was observed if fibroblasts were treated with OSKM and a compound that inhibits TGF-beta signaling or vitamin C. However, when all three of these compounds were combined, OSKM-engineered fibroblasts were reprogrammed at an efficiency of close to 80 percent. When different cell types were used as the starting cell, such as blood progenitor cells, the efficiency jumped to close to 100 percent; a result that was also observed if liver progenitor cells were used as the starting cell.

Stadtfeld is confident that these dramatic increases in iPSC derivation should improve future studies with iPSCs, since his protocol should make iPSC derivation more predictable. “It’s just a lot easier this way to study the mechanisms that govern reprogramming, as well as detect any undesired features that might develop in iPSCs,” he said.

Vitamin C and the two compounds used to manipulate the Wnt and TGF-β pathways have been widely used in research and have few unknown or hazardous effects. However, OKSM has in some cases caused undesired features in iPSCs, such as increased mutation rates. Stadtfeld believes that by making iPSC induction more rapid and efficient, his new technique might also make the resulting stem cells safer. “Conceivably it reduces the risk of abnormalities by smoothening out the reprogramming process,” Dr. Stadtfeld says. “That’s one of the issues we’re following up.”

Mesenchymal Stem Cells Make Tendons on Fabricated Collagen

Ozan Akkus and his colleagues from the Department of Mechanical and Aerospace Engineering at Case Western Reserve University in Cleveland, Ohio has succeeded in making fibers made completely from the protein collagen. Why is this a big deal? Because it is so bloody hard to do.

In a paper published the journal Advanced Functional Materials, Akkus and others describe the generation of their three-dimensional collagen threads. This is the first time anyone has described the formation of such threads made purely from collagen.

Collagen is a very widely distributed protein in our bodies. It is the major structural component of tendons, and most connective tissues, and as a whole, collagen composes approximately one-third of all the protein in our bodies. There are almost 30 different types of collagen; some collagens for stiff fibers and others form flat networks that act as cushions upon which cells and other tissues can sit.

Collagen biosynthesis is very complicated and occurs in several steps. First, the collagen genes are transcribed into messenger RNAs that are translated by ribosomes into collagen protein. However, collagen proteins are made in a longer, inactive form that must undergo several types of modifications before it is usable.

Collagen synthesis begins in a compartment of the cell known as the endoplasmic reticulum, which is a series of folded membranes associated with the nuclear membrane. Within the endoplasmic reticulum, the end piece of the collagen protein, known as the signal peptide, is removed by enzymes called signal peptidases that clip such caps off proteins. Now particular amino acids within the collagen protein chains are chemically modified. The significance of these modifications will become clear later, but two amino acids, lysine and proline, and -OH or hydroxyl groups added to them. This process is called “hydroxylation,” and vitamin C is an important co-factor for this reaction. Some of the hydroxylysine residues have sugars attached to them, and three collagen protein chains now self-associate to form a “triple ɣ helical structure.” This “procollagen” as it is called, is shipped to another compartment in the cell known as the Golgi apparatus. Within the Golgi apparatus, the procollagen it is prepared to be secreted to the cell exterior. Once secreted, collagen modification continues. Other proteins of the collagen protein chains called “registration peptides” are clipped off by procollagen peptidase to form “tropocollagen.” Multiple tropocollagen molecules are then lashed together by means of the enzyme lysyl oxidase, which links hydroxylysine and lysine residues together in order to form the collagen fibrils. Multiple collagen fibrils form a proper collagen fiber. Variations on a theme are also available, since collagen can also, alternatively, attached to cell membranes by means of several types of proteins, including fibronectin and integrin.


Now, if the cells has to go through all that just to make a collagen fiber, how tough do you think it is to make collagen fiber in a culture dish? Answer – way hard. In order to make collagen threads, Akkus and his team had to use a novel method for mature collagen production, and then they compacted the collagen molecules by means of the mobility of these molecules in an electrical field. This “electrophoretic compaction” method also served to properly align the collagen molecules until they formed proper collagen threads. Biomechanical analyses of these fabricated collagen threads showed that they had the mechanical properties of a genuine tendon. Akkus’ group when one step further and showed that a device they designed with movable electrodes could fabricate continuous collagen threads (). Thus, Akkus and his crew showed that they could make as many collagen threads as they needed and that these threads worked like tendons (see here for video). Are these guys good or what?

A. Schematic of basic electro-chemical cell layout for collagen alignment; B. Polarized image confirming the alignment of ELAC; C. Human mesenchymal stem cells on ELAC threads at day 1 and day 14. Cell form a confluent layer on day 14. Scale bar: 0.5 mm.
A. Schematic of basic electro-chemical cell layout for collagen alignment; B. Polarized image confirming the alignment of ELAC; C. Human mesenchymal stem cells on ELAC threads at day 1 and day 14. Cell form a confluent layer on day 14. Scale bar: 0.5 mm.

Nest, Akkus and his gang seeded collagen threads with mesenchymal stem cells (MSCs) from bone marrow. Remarkably, these collagen thread-grown mesenchymal stem cells differentiated into tenocytes, which are the cells that made tendons. Normally, MSCs do not readily form tenocytes in the laboratory, and they do not easily make tendons. However, in this case, the MSCs not only differentiated into tenocytes and made tenocyte-specific proteins and genes, but they do so without the addition of exogenous growth factors; the collagen threads were all the cells needed.

The seeded MSCs made Collagen I, which is the most abundant collagen of the human body, and is present in scar tissue, tendons, skin, artery walls, corneas, the endomysium surrounding muscle fibers, fibrocartilage, and the organic part of bones and teeth. Other tendon-specific proteins that were made included tenomodulin, and COMP (Cartilage oligomeric matrix protein). Furthermore, the electrically-aligned collagen does a better job of inducing the tenocyte fate in MSCs than collagen that is randomly oriented.

These remarkable and fascinating results demonstrate scaffolds made of densely compacted collagen threads stimulates tendon formation by Mesenchymal stem cells. Thus electrically aligned collagen as a very promising candidate for functional repair of injured tendons and ligaments. Now it is time to show that this can work in a living creature. Let the preclinical trials commence!!