UTHealth Research Shows Modified Adult Stem Cells May Be Helpful In Spinal Cord Injury

Spinal damage paralyzes people and drastically changes their lives. Healing people with spinal cord injuries could restore motility to paralyzed people. However, this is a very complicated bit of treatment, and a deal of work needs to be done. A result that might bring us closer to that goal comes from the University of Texas. Researchers at the University of Texas Health Science Center at Houston have shown that in spinal cord-damaged rats, transplantation of genetically modified adult stem cells can help restore movement (published in the Feb. 24 issue of the Journal of Neuroscience).

Spinal cord injury causes “demyelination.” This 50-cent words simply means the destruction of the sheath that surrounds the long bits of that extend from neurons in the central nervous system. This sheath is composed of a protein called myelin, and this myelin sheath is produced by special cells called oligodendrocytes. Oligodendrocytes wrap around the axons of nerves and augment the conduction of nerve impulses through nerve cell axons. Without myelin sheaths, the nerves cannot send messages to make muscles move.

Qilin Cao, M.D.,and his colleagues discovered that transplanted adult stem cells called oligodendrocyte precursor cells (or OPCs) from the spinal cord could become mature oligodendrocytes. The new cells could form myelin sheaths and help restore electrical pathways through the spinal cord. This process, whereby oligodendrocytes make the myelin sheath is called “remyelination.”

Cao and his co-workers isolated oligodendrocyte precursor cells from adult spinal cord and before they transplanted them, they genetically modified them to express a special protein called ciliary neurotrophic factor (CNTF), a protein that encourages nerve growth. CNTF facilitates survival and differentiation of Oligodendrocyte Precursor Cells in cell culture.  Perhaps the most important result is that the demyelination coincided exactly with the anatomical location where they were needed.

This study confirms stem cell grafting in attempts to remyelinate an injured spinal cord is a viable therapeutic strategy.  Secondly, it also shows that recovery regenerative treatments will require more than simply grafting naïve precursor cells.

Embryonic Stem Cells Restore Sight in Blind Mice

Retinitis pigmentosa is a group of genetic diseases that affect sight. The disease is progressive, usually beginning with night blindness, then proceeding to night blindness and then complete blindness. The progression of the disease is somewhat slow, and many people retain some sight for the remainder of their lives, but others become completely blind.

Retinitis pigmentosa is caused by the death of retinal photoreceptor cells. The abnormalities originate in either the photoreceptors themselves, or the retinal epithelium behind the photoreceptors into which the photoreceptors are embedded.

Is there a way to replace dead photoreceptors? Yes there is, at least in mice. An international research team has used mouse embryonic stem cells to replace diseased retinal cells and restore sight in a mice with retinitis pigmentosa. The team, led by scientists at Columbia University Medical Center, made retinal cells and used them to replace the dead photoreceptors. They suggested that this regenerative strategy could potentially become a new treatment for retinitis pigmentosa, which is a leading cause of blindness that affects approximately one in 3,000 to 4,000 people, or 1.5 million people worldwide. This study will appear in the journal Transplantation in the March 27, 2010 print issue.

The retinal pigment epithelium cells are specialized retinal cells and they help maintain vision.  Retinitis pigmentosa results from the death of these retinal cells on the periphery of the retina, which leads to “tunnel vision.”  In people with tunnel vision, the field of vision is narrowed considerably and everything outside the “tunnel” appears blurred or wavy.

In this study, sight was restored in one-fourth of the mice that received the stem cells, but complications of benign tumors and retinal detachments were seen in some of the mice.  Therefore there is a need to optimize these techniques to decrease these complications.  One modification that might be to use cells other than embryonic stem cells that do not cause tumors.

For example, inside the eye and lining the back part of it is a layer of photoreceptors that detect light, color, and images.  These photoreceptors convey this information to the brain with the help of several cells and assist them.  Collectively, this layer is called the retina.  Adjacent to the retina is a structure called the ciliary epithelium, which also harbors a stem cell population that divides and grows in response to retinal injury.  Because of this, ciliary epithelium stem cells are also called retinal stem cells or RSCs.  When grown in the laboratory, RSCs can differentiate into many of the cell types found in the retina (T.A. Reh and A. J. Fischer, Methods in Enzymology 2006;419:52-73).  Discovery of this stem cell population offers the possibility of using them to repair retinal damage.

Because retinal damage and death are a major component of many diseases of the aged like macular degeneration and Stargardt disease, this procedure might provide some very exciting ways to treat blindness in older patients.

Neurons from Skin Cells

Can we make nerve cells from skin cells? The answer seems to be yes. Furthermore, it seems to be really easy to do.

Marius Wernig at the Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology at Stanford University School of Medicine had published a remarkable paper. They started from a pool of nineteen candidate genes, but they identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that rapidly and efficiently convert mouse embryonic and adult mouse fibroblasts into functional neurons in vitro. He called these cells induced neuronal (iN) cells. He further showed that they expressed multiple neuron-specific proteins, and were able to generate nerve impulses and form functional connections between other nerve cells (synapses). Because they could make iN cells from non-nerve cells, they might be able to make large quantities studies of neurons for research.

Of even greater importance is the ability to make nerve cells for regenerative medicine. While it is too early to get too excited about this, the ability to form neurons from your own skin cells to repair spinal cord injuries and other neurological disorders without killing embryos is thrilling to say the least.

Reprogramming for Stem Cells

Regenerative medicine possesses tremendous potential. At the center of regenerative medicine is stem cells. How we derive stem cell lines is a central concern of this blog, but I remain convinced that embryonic stem cells do not represent the future of regenerative medicine. My reasons are manifold, but one of my greatest concerns is that embryonic stem cells (ESCs) require the death of human embryos. Human embryos are young human persons at the earliest stages of life. Destroying them is killing an innocent person. There has to be a better way.

Induced pluripotent stem cells (iPSCs) provide one possible alternative to ESCs, and while these cells show tremendous promise, they have their share of problems. While many of the safety concerns with these cells have been nicely addressed, others remain. Is there an even better way?

Hopefully the answer is “yes.” As it turns out, it is possible to reprogram cells to form another cell type without taking them through an embryonic-like stage. This strategy is called reprogramming, and it has been used by Doug Melton and co-workers in his lab at Harvard University to make insulin-making beta cells from other types of pancreas cells that do not normally make insulin (Qiao Zhou, et al., Nature 455, 627-632).  Likewise, the steroid dexamethasone can convert pancreatic cells into liver cells.

Now other researchers have found that small molecules can reprogram cells to become another cell type.  Small molecules can cause unwanted side effects, but James Chen, a chemical biologist at Stanford University School of Medicine, says they “are more in our comfort zone in terms of clinical therapies.”  Chen also said, “Chemists can synthesize and derivatize them, there are standard methods for determining compound pharmacokinetics, and the path to FDA [Food & Drug Administration] approval is well established.”

Researchers also favor small molecules because they have more control over dosage and delivery time with them than they do with genetic techniques.

In 2007, Sheng Ding, chemical biologist at Scripps Research Institute, reported the first small molecule that could substitute for one of the four reprogramming transcription-factor genes. Researchers continue to identify small molecules that can replace one, two, or three of the four reprogramming factors. Among the newest transcription-factor gene stand-ins are molecules such as the lactam kenpaullone from Peter Schultz’s laboratory at Scripps and the heterocyclic RepSox from Lee Rubin and Doug Melton at Harvard Stem Cell Institute (Proc. Nat. Acad. Sci. USA 2009, 106, 8912; Cell Stem Cell 2009, 5, 491).

Reprogramming might be able to do great things.  Out bodies are filled with stem cells.  We just need to know how to manipulate them.

Induced Pluripotent Stem Cells do not Form Neural Stem Cells as well as Embryonic Stem Cells

Induced pluripotent stem cells show tremendous promise for regenerative medicine. However in a February 15th article in the Proceedings of the National Academy of Sciences, showed that induced pluripotent stem cells (iPSCs) were inefficient at forming the cells of the brain in comparison to their embryonic stem cell counterparts.

The senior author of the article , Su-Chun Zhang, (professor, University of Wisconsin-Madison School of Medicine and Public Health), said:  “Embryonic stem cells can pretty much be predicted,” and “Induced cells cannot. That means that at this point there is still some work to be done to generate ideal induced pluripotent stem cells for application.”

This study compared the ability of five different embryonic stem cell lines to 12 different iPSC lines to form nerve cell precursors.  Embryonic stem cells are considered the “gold standard” for all pluripotent stem cells, which are cells that can differentiate into all of the 220 cell types in the human body.  Zhang’s group found that the induced cells differentiated into progenitor neural cells and further into the different kinds of functional neurons that make up the brain, but they did not faithfully reproduce all the differentiation capabilities of embryonic stem cells.  This suggests that there are unknown factors at play that may limit the use of iPSCs when it comes to modeling diseases in the laboratory.  Such unknowns would also limit their use in clinical settings for such things as cell transplants.

Despite their unpredictability, Zhang notes that iPSCs can still be used to make pure populations of specific types of cells, which makes them useful for some applications like testing potential new drugs for efficacy and toxicity.  Zhang also noted that the limitations identified by his group are technical issues likely to be resolved relatively quickly.  “It appears to be a technical issue,” said Zhang.  “Technical things can usually be overcome,” he added.

This is very possibly a technical issue that is due to our inability to properly manipulate iPSCs to form nerve cells.  However, if the same protocols that drive embryonic stem cells to form nerve cells are used on iPSCs, they only form nerve cells poorly.  There are probably other protocols that can do just this.  We just haven’t found them yet.

Also, it is worth mentioning, that the ability of iPSCs to differentiate into neurons is probably a line-specific property.  Therefore if these lines to not form lines effectively, then perhaps other lines do.

Induced Pluripotent Stem Cells Improve Hearts

Mayo Clinic Investigators have shown that induced pluripotent stem cells, which were made from particular cells in the skin called “fibroblasts,” were differentiated into heart muscle cells and used to treat mice with heart disease. This proof-of-principle study shows that is might be possible to use iPSCs to fix hearts after a heart attack with iPSCs.

Timothy Nelson, the principal author of this study, said that this study “establishes the real potential for using iPS cells in cardiac treatment. iPSCs have already been used to treat sickly cell anemia, Parkinson’s disease and hemophilia A in laboratory mice. This experiment, which was also done in mice, further extends the clinical conditions that iPSCs might treat.

This is an exciting result, but there is a caveat I must mention. Christine Mummery and her colleagues have shown that even though human embryonic stem cell transplantation improves the condition of the heart after a heart attack in rodents after four weeks, examination of these same rodents twelve weeks after the transplants reveals that the improvements have largely disappeared.  In this study, the mice were examined after four weeks and not after twelve. Therefore, this study might be in the same category as those done with human embryonic stem cells. If the improvements could be shown to last even up to twelve weeks after the transplantations, then I think we would have something really to crow about. However, as it is, while this result is interesting, it is simply not conclusive.

Using Plasmids to make induced pluripotent stem cells

Induced Pluripotent Stem Cells (iPSCs) are made from normal body cells. Normal body cells are engineered with specific genes that force the cells into a “more primal” or less differentiated state. These cells behave very much like embryonic stem cells (ESCs). Although there are some differences between iPSCs and ESCs, they are similar enough to each other to suggest that iPSCs can replace ESCs as the great hope for regenerative medicine.

A major concern of IPSCs is the manner in which they are made. Body cells are infected with recombinant retroviruses and these retroviruses introduce the genes necessary to transform the body cells into iPSCs. However, retroviruses tend to insert genes into the human genome, largely at random, and this can produce mutations that can kill cells or even convert them into cancer cells. To deal with this problem, scientists have tried to produce iPSCs with other strategies.

First, scientists have used viruses that do not insert genes into the genome. Adenoviruses, for example, do not insert genes into the genome of the cells they infect, and once the infection is complete they, in this case, do not persist. Researchers have used engineered adenoviruses to convert body cells into iPSCs (Matthais Stadfeld et al., Science 322 (2008): 945-9). Other researchers have even found ways to convert body cells into IPSCs without even using viruses (Keisuke Okita et al., Science 322 (2008): 949-53; Knut Woltjen et al., Nature 458 (2009): 766-70). Also, scientists have found a way to use retroviruses that self-destruct after they have inserted into the genome (Keisuke Kaji et al., Nature 458 (2009): 771-5).

A new report now shows that it is possible to make iPSCs by using genes that are loaded into small circles of DNA. Michael Longaker, professor of surgery at Stanford University, used “minicircles” of DNA that did not contain any bacterial DNA to reprogram human fat cells into iPSCs.  The senior author of this research, Joseph Wu, said, “Imagine doing a fat or skin biopsy from a member of a family with heart problems, reprogramming the cells to pluripotency and then making cardiac cells to study in a laboratory dish.”  He continued, “This would be much easier and less invasive than taking cell samples from a patient’s heart.”  This article was published online Feb. 7 in Nature Methods.

This protocol works quite well because the minicircle vector with the reprogramming genes on it quite small; it only contains the four genes needed to reprogram the cells (plus a gene for a green fluorescent protein to track minicircle-containing cells).  The expression of the reprogramming genes on the minicircles is quite robust, and the smaller size of the minicircles allows them to enter the cells more easily than the larger segments of DNA.  Also, these minicircles do not replicate and are naturally lost as the cells divide.  Therefore they do not remain in the cell and cannot cause any later problems.

These researchers chose to use fat cells because they are numerous, easy to isolate and amenable to the iPS transformation.  In this work, they found that about 10.8 percent of the stem cells took up the minicircles and expressed the green fluorescent protein, or GFP, versus about 2.7 percent of cells transformed by more traditional means.

It gets better though.  Isolation of the GFP-expressing cells showed that the minicircles were gradually lost over a period of four weeks.  After a second dose of the minicircles 4 to 6 days  and after 14 to 16 days, they observed clusters of cells resembling embryonic stem cell colonies – some of which no longer expressed GFP.

This new procedure is a remarkable advance towards safety for iPSCs and places them a step close to being used in therapeutic trials.

Mesenchymal stem cells form heart muscle

On August 3rd, 2009, the University of Miami Miller School of Medicine released a press piece that reported the results on a study by Joshua M. Hare, who is the director of the Interdisciplinary Stem Cell Institute at the Miller School. This study examined the ability of mesenchymal stem cells to fix ailing hearts.

Mesenchymal stem cells are found in lots of different places in our bodies. They are found in bone marrow stroma, fat, connective tissue, blood vessels, umbilical cord, and lots of other places too. These cells might come from “perivascular” cells, which are cells that hang around blood vessels. Nevertheless, mesenchymal stem cells have the ability to form bone, cartilage, fat, and muscle. They also have a fascinating capacity to hide from the immune system. They have groups of surface proteins that prevent cells from the immune systems from recognizing them as foreign, and therefore, mesenchymal stem cells from one person can be transferred into an unrelated person without fear of transplantation rejection.

Several experiments have shown that mesenchymal stem cells (MSCs) can differentiate into heart muscle if treated with the right chemicals (S. Tomita, et al., Circulation 1999;100:II-247–II-256; Also see H. Okura, et al., Tissue Eng Part C Methods, 2009). Transplanting MSCs into the hearts of laboratory animals that have had heart attacks can also help the fix the heart (D. Wolf, et al., J Am Soc Echocardiogr 2007;20:512-20). However, there is a raging debate over how MSCs help broken hearts get better.

Even though MSCs can form heart muscle in culture, they seem to do so rather poorly (Y. Zhang, et al., Interact Cardiovasc Thorac Surg. 2009 Dec;9(6):943-6). Also, several studies suggest that once MSCs are transplanted into ailing hearts, they do not differentiate into heart muscle with any efficiency worth bragging about and seem to help the heart by means of the chemicals they produce (Ryota Uemura, et al., Circulation Res 98 (2006): 1414-21).

There are, however, some reasons to suspect that this is not the end of the story. Engineering MSCs with various genes or administering MSCs with certain chemicals can push then to form heart muscle at higher rates (Yigang Wang, et al. Am J Physiol Heart Circ Physiol (nov 6, 2009, doi:10.1152/ajpheart.00765.2009). Also, in particular experiments, MSCs clearly form heart muscle (J. Tang, et al., Eur J Cardiothorac Surg 30 (2006): 353-61).

Clinical studies with MSCs for heart problems have been conducted but the data are limited. Initial studies were very encouraging (S. Chen, et al., Am J Cardiol 94 (2004): 92-5 and S. Chen, et al., J. Invasive Cardiol 18 (2006): 552-6). Now a new study has shown that MSCs not only help people who have had a recent heart attack, but that they turn into heart muscle and other heart tissues.  MSCs can also help form blood vessels and the increase of blood flow to the heart also helps an ailing heart.  This seems to be one of the main ways that bone marrow-based stem cells help hearts after a heart attack.  Therefore MSCs might be one of the best ways to treat bum hearts, but certainly more work needs to be done.