Proteins as gene carriers

By now you probably know everything about pluripotent stem cells, right? They are the hot topic in genetics right now, to the point that the fear of being scooped has pushed some people to lie about their results. Pluripotent stem cells are cells that have the ability to divide into a specialized cell and another undifferentiated cell. This of course is greatly useful in repairing damaged organs and/or regenerating tissue, and has great potential in medicine.

Lately there has been a lot of buzz on the notion that pluripotency could be re-induced in already differentiated cells. Studies have shown that four reprogramming factors can indeed reprogram fibroblast cells into pluripotent stem cells when over-expressed.

But how to over-express these factors?

The typical route is to transfect the genes into the cells by means of a viral vector. Basically, the genes are delivered into the cell using a retrovirus. Though effective, this poses the question of side effects: whenever you introduce foreign DNA into a cell you have the potential to silence secondary genes or disrupt the usual gene regulation. Unanticipated epigenetic changes in the cell can occur. A recent study [1] shows a safer alternative: cell-permeant proteins, or CPPs. These are small proteins that can cross the cell membrane and carry peptides inside the cell in a process called "protein transduction," thus offering a valid alternative to viral vectors.

By comparing the two methods (CPPs and viral delivery) on human fibroblast cells, Lee et al. noticed that gene expression was achieved much faster when using the viral vector. Puzzled by this difference, they wondered what was so special about the viral route that made the gene delivery so much more efficient. There had to be something in the viral vector that aided the delivery of the genes. Lee et al. hypothesized that this could be linked to the fact that the viral vector somehow activated an inflammatory pathway in the cells which in turn aided the delivery of the genes. So the next question was: can we enrich the CPPs so they too activate the inflammatory pathway?

Indeed they could! They used TLR3 agonists, molecules that activate the TLR3, or Toll-like receptor 3, a receptor that recognizes double-stranded RNA generated by retroviruses and thus activates inflammatory pathways. Once combined with the TLR3 antagonists, over-expression of the reprogramming factors was achieved faster through CPPs than it was with the viral vectors, validating the hypothesis that the gene delivery has to be achieved via the activation of the immune pathway. In fact, the contrary was also true: when TLR3 was knocked down (biology jargon to say that the gene was silenced), the viral vector was also inefficient in delivering the genes.

"TLR3 activation enables epigenetic alterations, including changes in methylation status of the Oct4 and Sox2 promoters as well as changes in the expression of epigenetic effectors, that promote an open chromatin configuration. The knowledge that the activation of innate immune response affects nuclear reprogramming permitted us to enhance the efficiency and yield of human induce pluripotent stem cells by using reprogramming factors in the form of CPPs."

Lee et al. conclude:

"Our observations highlight a previously unrecognized role for innate immunity activation in nuclear reprogramming. The viral vectors constructs used to induce pluripotency are more than mere vehicles for the reprogramming factors. Innate immune activation causes striking changes in epigenetic modifiers that favor an open chromatin configuration. These changes enable a fluidity of cell phenotype that contributes to successful nuclear reprogramming."

[1] Lee, J., Sayed, N., Hunter, A., Au, K., Wong, W., Mocarski, E., Pera, R., Yakubov, E., & Cooke, J. (2012). Activation of Innate Immunity Is Required for Efficient Nuclear Reprogramming Cell, 151 (3), 547-558 DOI: 10.1016/j.cell.2012.09.034

Lorenzo's oil got upgraded to stem cell research

Have you seen the 1992 movie Lorenzo's oil? The film portrays the true (and sad!) story of Lorenzo Odone, who, at age 6, was diagnosed with adrenoleukodystrophy, one of the most common forms of leukodystrophies, a family of degenerative diseases that affects the growth of the myelin sheath. Myelin wraps around nerve fibers creating a fatty covering that increases the speed at which impulses propagate. Leukodystrophy is a genetic disorder caused by mutations in the genes that code myelin proteins. When myelin is defective, or not produced in sufficient quantities, it starts degrading, causing the progressive loss of signaling along the nerve. Eventually, the nerve dies.

As shown in the movie Lorenzo's oil, Lorenzo's parents refused to accept the common prognosis they were given at the time for their son (progressive paralysis and death within 2-3 years). Their determination led them to discover an oil mix able to alleviate the symptoms of the disease.

Two papers published in Science Translational Medicine now show that stem cell therapy can partially regenerate neurological function.

Uchida et al. [1] show that stem cell transplantation is effective in the regeneration of the myelin sheath in mouse models. The researchers transplanted human central nervous system stem cells (HuCNS-SCs) into the brains of mice with defective myelination in the central nervous system. The transplanted stem cells generated functional myelin in the mice's central nervous system

In the same issue, Gupta et al. [2] describe how they transplanted the same cells (HuCNS-SCs) into the frontal lobe of four young boys that were affected by Pelizaeus–Merzbacher disease (PMD), a form of leukodystrophy. The transplant was followed by a 9-month regimen of immunosuppression to minimize the chances of rejection. One year after the transplant, magnetic resonance imaging (MRI) showed that the transplanted cells had engrafted and successfully myelinated brain cells. The researchers conclude that "modest gains in neurological function were observed in three of the four subjects. No clinical or radiological adverse effects were directly attributed to the donor cells."

Sadly, Lorenzo died in 2008, one day after his thirtieth birthday. His story, though, was and still is an inspiration to many.

[1] Uchida, N., Chen, K., Dohse, M., Hansen, K., Dean, J., Buser, J., Riddle, A., Beardsley, D., Wan, Y., Gong, X., Nguyen, T., Cummings, B., Anderson, A., Tamaki, S., Tsukamoto, A., Weissman, I., Matsumoto, S., Sherman, L., Kroenke, C., & Back, S. (2012). Human Neural Stem Cells Induce Functional Myelination in Mice with Severe Dysmyelination Science Translational Medicine, 4 (155), 155-155 DOI: 10.1126/scitranslmed.3004371

[2] Gupta, N., Henry, R., Strober, J., Kang, S., Lim, D., Bucci, M., Caverzasi, E., Gaetano, L., Mandelli, M., Ryan, T., Perry, R., Farrell, J., Jeremy, R., Ulman, M., Huhn, S., Barkovich, A., & Rowitch, D. (2012). Neural Stem Cell Engraftment and Myelination in the Human Brain Science Translational Medicine, 4 (155), 155-155 DOI: 10.1126/scitranslmed.3004373

Reprogrammable cells

Can't remember if I already shared the above picture... it's my favorite sunset shot so far, so forgive me if it's a deja vu.

The Nobel Prize in medicine this year was awarded to John Gurdon and Shinya Yamanaka for pioneering the reprogramming of cells into an embryonic-like state. Embryonic stem cells are cells that undergo asymmetric division, as they divide into an undifferentiated cell and into a specialized cell. This way, they can grow indefinitely while maintaining their undifferentiated state and, at the same time, keep the ability to differentiate into all three germ layers, the cells formed during embryogenesis.

When still a PhD student, in 1958, Gurdon cloned a frog using the nucleus of a cell taken from the intestine of a tadpole. It took another 38 years before the first mammal was cloned: the first cloned sheep, Dolly, was born in 1996 from an unfertilized egg whose nucleus had been replaced with the nucleus of an adult cell. In this case, the adult cell, by being placed into the egg, was effectively "reprogrammed" into an embryonic stem cell. Up until Gurdon's work was published in 1962, general belief was that once cells specialized, they could not revert. The discovery that cells can actually undergo "reprogramming" under special circumstances is quite significant because it gives hope that we can achieve tissue regeneration and treat degenerative diseases or spinal cord injuries.

In 2006 Yamanaka and his colleague Kazutoshi Takahashi published a paper in Cell [1] in which they showed that, activating four genes, they were able to reprogram adult fibroblasts from mouse embryonic cells. They called the new cells induced pluripotent cells, or iPS, and found that they expressed embryonic-state cell markers. In fact, once in the proper environment, they contributed to embryonic development.

Yamanaka is a strong believer that this research will eventually lead to successful regeneration therapies. In fact, he plans to start a bank of induced pluripotent stem cells obtained from 75 different cell lines. Is this the beginning of a new era? A word of caution comes from a paper published in PNAS at the end of 2010 [2]: in this paper, Serwold and colleagues derived mice from reprogrammed T-cells (cells from the immune system) and showed that roughly half of the mice generated this way spontaneously developed T-cell lymphomas.

The mice were generated by transferring T-cell nuclei into enucleate oocytes. As they mature, T-cells undergo genomic rearrangements, and while normally these rearrangements occur in T-cells only during a specific stage of their development, such rearrangements were observed in all somatic cells in the cloned mice. In [2] Serwold et al. show that these rearrangements undergo T-cell lymphomagenesis: in other words, they cause cancer. Though T-cells are not the only cells that currently can be reprogrammed, this study clearly shows that different cell lines can yield different outcomes, some quite deleterious.

"This study suggests that precautions should be taken to ensure that the identity of the reprogrammed cell of origin is known, and that T cells, and probably also B cells, are not inadvertently turned into therapeutic iPS cells. Recent studies have used human blood-derived T cells as sources of iPS cells, and these cells promise to be valuable tools for studying human immune development and disease; however, the results presented here indicate that extra caution is warranted regarding the therapeutic use of such T cell-derived iPS cells [2]."

[1] Takahashi, K., & Yamanaka, S. (2006). Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors Cell, 126 (4), 663-676 DOI: 10.1016/j.cell.2006.07.024

[2] Serwold, T., Hochedlinger, K., Swindle, J., Hedgpeth, J., Jaenisch, R., & Weissman, I. (2010). T-cell receptor-driven lymphomagenesis in mice derived from a reprogrammed T cell Proceedings of the National Academy of Sciences, 107 (44), 18939-18943 DOI: 10.1073/pnas.1013230107

Limb regeneration: a lesson from salamanders

As much as we would love to enlist limb regeneration among modern science's best accomplishments, so far it is still very much confined to science fiction. That doesn't mean it won't happen, though. Key to limb regeneration is cellular reprogramming that allows differentiated cells to return to a germline-like (undifferentiated) state. Genes involved in embryonic development need to be reactivated in order to restart the same process that created the limb during the growth of the embryo.

The vertebrates with the best ability to regenerate limbs are salamanders, making these little critters the most studied in the field. When a salamander loses a limb, a layer of epidermis grows to cover the wound, and beneath this layer new, undifferentiated cells start proliferating, forming a mass called blastema. Recent research shows that this first wave of cell dedifferentiation may recapitulate events occurring during embryogenesis.

"The cells in the limb blastema are believed to be a heterogeneous collection of dedifferentiated cells that have been reprogrammed to achieve varying levels of developmental potential exhibited by the cells involved in embryogenesis [1]."

Germline stem cells are cells that give rise to gametes (the reproductive cells) and have the ability to divide into another stem cell as wells as a more differentiated cell. This mechanism, called asymmetric division, is controlled by a protein called PIWI through small, non-coding RNAs called piRNAs. In [1] Zhu et al. showed that when salamanders regenerate a limb, a germline-like state is established in the growing tissue. In particular, they found that germline-specific genes were expressed in the regenerated limb. In order to show this, they looked specifically at the PIWI proteins.

Zhu and colleagues found a significant amount of upregulated transposable elements in the regenerated limbs. If you remember, transposable elements is a segment of DNA that can move from one locus to another within the genome of the same cell. During the limb regeneration process, transposable elements can impart a deleterious amount of instability, which is counteracted by a corresponding upregulation of the PIWI genes. Conversely, when the PIWI genes were knocked down (i.e. their expression was reduced) in the blastema, limb growth following the amputation was significantly reduced compared to controls.

Zhu et al. conclude

"In the future, further characterization of the subpopulations of these reprogrammed cells with additional germline-specific markers might provide more insight into exactly how far cellular dedifferentiation can proceed and whether there are indeed a small number of cells that could be isolated before a certain developmental threshold and exhibit true pluripotency when isolated from the influence of the partially programmed blastemal cells in the proximity."

[1] Wei Zhu, Gerald M. Pao, Akira Satoh, Gillian Cummings, James R. Monaghan, Timothy T. Harkins, Susan V. Bryant, S. Randal Voss, David M. Gardiner, & Tony Hunter (2012). Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl Developmental Biology DOI: 10.1016/j.ydbio.2012.07.021

Regenerating tissue through autologous cells: a personal appeal

The trachea is one of the most challenging organs to transplant, with a high risk of necrosis and infection due to inadequate graft revascularization and the fact that it's constantly exposed to airborne elements. Transplants requires lifelong immunosuppression, which also carry high risks. Prosthesis can rupture, generate infection, and cause injury.

What to do then? One answer is tissue engineering.

Dr. Paolo Macchiarini is one of the pioneers in this techniques. In a recent paper [1] he and his co-authors

"describe in detail the tissue engineering approach used for tracheal construction, with a focus on the mobilization, isolation, and in vitro culture of cell types with high potential for use in bioengineering."

The technique is highly sophisticated and I'm sure I'm doing a poor job here in trying to explain it in simple terms. The starting point is a scaffold that should provide the basic characteristics of the trachea. As Macchiarini and colleagues state in the paper,

"Despite intensive research in this field, no solution has been proposed as being optimal; currently both natural and synthetic grafts are being used."

In one case study in particular, they used as scaffold a decellularized cadaveric organ from a human donor trachea, and then colonized it by epithelial cells and MSC-derived chondrocytes cultured from autologous cells taken from the patient. They aspirated bone marrow from the patient to obtain marrow mononuclear cells. These contain a class of repair cells called multipotent mesenchymal stem/progenitor cells, cells that are able to differentiate and hence can be used to regenerate tissue. The researchers separated the cells, differentiated them, and then seeded them along a scaffold:

"We then expanded and differentiated these cells toward chondrocytes and seeded the cells into the exterior spongy layer of the scaffold, where they formed the cartilaginous component. For generating the inner epithelial lining of the trachea, we seeded the surface of the scaffold with nasal epithelial cells, after in vitro expansion to obtain sufficient numbers for seeding the graft."

Above: The entire concept of the regenerative approach to tracheal transplantation using natural scaffolds. MNC, mononuclear cell.

While ex-vivo, the tissue is maintained through a perfusion system called bioreactor. Once implanted, several pharmacologic intervention are prescribed to minimize the risk of necrosis, infection, and cell migration. Despite the non-trivial risks, the result is incredible:

"Since 2008, nine patients (ranging in age from 11 to 73 years), with either benign or malignant conditions, were treated using this decellularized scaffold. To date, the new in vivo engineered transplanted tracheas have been shown to be viable and to possess a good epithelial coating, are characterized by immediate vascularization, and, above all, maintain a constantly open lumen for air passage."

Please help Rachel Breathe

Now, to most of us, what I've discussed above is fascinating science. To some, is hope for a new life. Rachel Phillips was a ballet dancer with Royal Ballet in London, the Kirov in St. Petersburg, Russia and other major companies in the US and abroad. Today, with over 90% of her airways collapsed, Rachel is fighting for her life. She suffers from a genetic disorder called Ehlers–Danlos syndrome, which is caused by mutations in a number of genes involved in either the structure, the production, or the processing of collagen. Collagen is essential in all connective tissues in the body. Because of this Rachel needs a new trachea and Dr. Macchiarini's tissue regeneration technique can give her one but she needs our help.

Please visit Rachel's website at helprachelbreathe.com/ and help her out with a donation. This is not just science. It's life!

Thank you.

[1] Jungebluth, P., Moll, G., Baiguera, S., & Macchiarini, P. (2011). Tissue-Engineered Airway: A Regenerative Solution Clinical Pharmacology & Therapeutics, 91 (1), 81-93 DOI: 10.1038/clpt.2011.270