A research team at University of California San Francisco, has discovered how to genetically reprogram human cells without the use of viruses to insert DNA. The breakthrough technique has far reaching significance in medicine, research and industry. Using CRISPR gene editing technology in a versatile, rapid and economical approach, the team believes the technique will be adopted in the field of cell therapy which could accelerate the development of new and safer treatments for a variety of diseases.

The new method is a robust molecular “cut and paste” technique which was used to rewrite genome sequences in T cells. The technique relies on electroporation which is a process where an electrical field is applied to cells which makes their membranes temporarily more permeable. The UCSF team discovered that when certain quantities of T cells, DNA and CRISPR scissors were combined and then exposed to an appropriate electrical field, the T cells will absorb these elements and then integrate specific genetic sequences very precisely at the site of a CRISPR programmed cut in the genome.

The method is a rapid and flexible technique that can be used to enhance, reprogram and alter T cells which can be given specificity to a variety of diseases to tamp down excessive immune response. Just as important as the new technique’s ease of use and speed, the approach has made it possible to insert substantial stretches of DNA into T cells which will endow the cells with powerful new properties. Previously, some success had been achieved using electroporation and CRISPR to insert bits of genetic material into T cells, however not until the new research were they able to place long sequences of DNA into T cells without causing the cells to die leading them to believe DNA sequences were excessively toxic to T cells.

Through trial and error, the researchers determined the ratios of DNA quantity, T cell population and CRISPR abundance that when combined with an electrical field and delivered with proper parameters, the result was an accurate and efficient editing of the T cells genomes. To validate their findings, they directed CRISPR to label a variety of different T cell proteins with GFP (green fluorescent protein) and the outcome was very specific with very low levels of off target effects. T cells for this experiment were from three siblings with a rare and severe autoimmune disease. These children carry mutations in a gene called IL2RA.

To further serve as proof of principle of the new technique’s therapeutic promise, the team showed how it could possibly be used to marshall T cells against disease. Using a non viral CRISPR technique, the UCSF team were able to quickly repair the IL2RA defect in the children’s T cells and restore cellular signals that had been impaired by the mutations. Referred to as CAR-T therapy, T cells that have been removed from the body are engineered to improve their disease fighting abilities and then returned to the body.

In another set of experiments, the scientists were able to completely replace native T cell receptors in a population of normal human T cells with new receptors that had been engineered to find diseased cells. T cell receptors are cell sensors that detect disease or infection. In lab dishes the engineered cells were able to efficiently hone in on targeted disease cells while ignoring other cells which shows the sort of specificity that is a major goal in precision disease medicine. With the new technique, they are able to cut and paste into a specified place rewriting a specific page in the genome sequence.

The new technique has made is possible to create viable custom T cell lines in just over a week. Ideas for earlier experiments were deemed too expensive or difficult due to obstacles presented by viral vectors. Because they can create CRISPR templates very quickly, as soon as a template is created they can get it into T cells and grow them very rapidly which makes a variety of experiments now ripe for investigation.

To view the original scientific study click here: Reprogramming human T cell function and specificity with non-viral genome targeting

Researchers at the Stowers Institute for Medical Research have discovered the one cell that has the capability of regenerating an entire organism. Until recently scientists lacked the tools that could target and track this cell which enables a variety of creatures such as the planarian flatworm to perform amazing feats such as regrowing a severed head.

By pioneering a new technique that combines single cell analysis, flow cytometry, imaging and genomics, the researchers have isolated this regenerative cell which is a subtype of the earlier studied adult pluripotent stem cell before it performed this remarkable act. Now that this stem cell has been isolated prospectively the findings prove that this is no longer an abstraction. There really is a cellular entity that can restore to animals and humans the ability to regenerate any part of the body.

All multicellular organisms are built from a single cell which divides multiple times. Each of the cells has the exact same twisted strands of DNA and is considered pluripotent which means it can give rise to all cell type possibilities in the body. However somewhere along the way the starter cells which are known as embryonic stem cells find a different fate and become heart cells, muscle cells, skin cells and other types of cells. In humans no known pluripotent stem cells will remain after birth, however in planarians they remain into adulthood. Here they become known as adult pluripotent stem cells or neoblasts. It is believed these neoblasts contain the secret to regeneration.

It is only in the last 20 years that scientists have been able to characterize this powerful cell population using molecular techniques and functional assays. The work showed that this seemingly homogenous cell population was a conglomeration of different subtypes all with different patterns and properties of gene expression. Previous to the current study, scientists would have to transplant over a hundred single cells into the same amount of worms to find the one that is truly pluripotent and has the ability to regenerate the organism. Not only would that be a lot of work, but to define it molecularly by identifying the genes that cell is expressing they would have to destroy the cell for processing which meant not being able to keep the cell alive to track its regeneration.

The team began searching for a distinguishing characteristic that would identify this elusive cell ahead of time. A stem cell marker known as piwi 1 is able to distinguish neoblasts so the team decided to begin there. They first separated the cells that expressed this marker from the ones that did not. They then noticed the cells could be separated into two groups…one that expressed lower levels of piwi 1 and ones that expressed high levels of piwi 1. They found that the ones that were piwi 1 high were the ones that fit the molecular definition of neoblasts and so the other ones were discarded.

This type of gene expression and protein levels had never been conducted in planarians. Previously researchers believed all cells which expressed the piwi 1 were true neoblasts and it wasn’t relevant how much of the marker they expressed. The team showed it did make a difference.

The researchers selected 8,000+ of the high piwi 1 cells and proceeded to analyze their gene expression patterns. To their surprise, the cells fell into 12 different subgroups. Through elimination they excluded any subgroups that had genetic signatures indicating they were cells destined for a particular fate such as skin or muscle cells. That left them with two subgroups that could still be pluripotent. These two groups were called Nb1 and Nb2.

The cells in NB2 group expressed a gene coding for a member of the tetraspanin protein family which is a group of evolutionary ancient and also very poorly understood proteins that sit on the cell surface. They made an antibody that could latch onto this protein and pull the cells that carried it out of a mixture of other neoblasts. They then transplanted the single purified cell into a planarian which had been subjected to lethal levels of radiation. The cells not only repopulated and rescued the planarian, but they did so 14 times more consistently than cells which had been purified by older methods.

The fact that the marker the team discovered is expressed not only in planarians but also in humans, opens the door to new experiments never thought possible. It would make sense that these principles could be broadly applicable to any organism that has ever relied on stems cells to become what they are today. That is everybody!

To view the original scientific study click here: Prospectively Isolated Tetraspanin Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration

Researchers at the Perelman School of Medicine at the University of Pennsylvania along with the Plikus Laboratory for Development and Regenerative Biology at the University of California, Irvine, have discovered a way to heal wounds without scars by manipulating woulds to heal as regenerated skin. Previously thought to be impossible, the researchers have found a way to transform cells found in wounds into fat cells.

Adipocytes which are fat cells are typically found in the skin but they become lost when wounds heal as scars. Myofibroblasts are the most common cells found in wound healing which were thought to only form a scar. Scar tissue will not have hair follicles or fat cells which is what gives them their abnormal appearance. Using these characteristics, the researchers had the basis for their work which was comprised of changing the present myofibroblasts into fat cells which do not cause the typical scarring as the result of wound healing. The method was to regenerate hair follicles first then the fat would regenerate in response to those signals.

The study showed that fat and hair develop separately but not independently. Hair follicles will form first and the study discovered factors that were necessary for their formation. These additional factors were actually produced by the regenerating hair follicle converting surrounding myofibroblasts to regenerate as fat in place of a scar. This fat will not regenerate without the new hairs and once it does the new cells are indistinguishable from the existing fat cells giving the wound a more natural appearance.

The researchers identified a factor called Bone Morphogenetic Protein that sends the signal from the hair to the fat cells, instructing the myofibroblasts to turn into fat. This discovery alone was groundbreaking as it changed what was previously known about myofibroblasts. They were thought to be incapable of turning into a different type of cell. The study showed that they have the ability to influence cells and can be efficiently and stably converted into adipocytes. This transformation was shown in both mouse and human keloid cells which were grown in culture.

The discoveries show a great opportunity for wound healing to regenerate tissue rather than scarring. From a clinical standpoint the results are highly desirable and have the potential to revolutionize dermatology. Right now it is an unmet need.

The study also showed that the increase of fat cells in tissue might also be helpful for more than wound healing. Adipocyte loss is a fairly common complication of other conditions such as treatments for HIV and for the aging process when cells are lost naturally which leads to permanent deep wrinkles and discolouration.

To view the original scientific study click here: Regeneration of fat cells from myofibroblasts during wound healing