Finding the Optimal Spot for Stem Cell Injections In Spinal Cord Injured Patients

A gaggle of laboratory animal experiments and clinical studies in human patients have established that stem cell injections into the spinal cord after spinal cord injury promote functional recovery (see Beattie, M. S., et al., Exp. Neurol. 148(2):453‐463; 1997; Bennett, D. L., et al., J. Neurosci. 20(1):427‐437; 2000; Kim HK, et al., PLos One 4(3): e4987 2009; Lu, P.; Tuszynski, M. H. Exp. Neurol. 209(2):313‐320; 2008; McTigue, D. M., et al., J. Neurosci. 18(14):5354-5365; 1998; Widenfalk, J.; Lundströmer, K. J. Neurosci. 21(10):3457‐3475; 2001; also see Salazar DL, et al., PLoS ONE, August 2010; Hooshmand M, et al., PLoS ONE, June 2009; Cummings BJ, et al., Neurological Research, July 2006; and Cummings BJ, et al., PNAS, September 19, 2005).  Stem Cell, Inc., for example, has conducted several tests with human patients using their HuCNS-SC human neural stem cell line, and transplantation of these stem cells promotes functional recovery in human patients who have suffered spinal cord injury.

However, one factor that has yet to be properly determined is the best site for stem cell injection. Previous work by scientists at the Keio University School of Medicine in Japan has shown that injection of neural stem cells and neural progenitor cells (NS/PCs) into non-injured sites by either intravenous or intrathecal (introduced directly into the space under the arachnoid membrane of the brain or spinal cord) administration failed to produce sufficient engraftment of stem cells at the site of injury.

Arachnoid space

Instead cells were trapped in the lungs and kidneys, and many mice even developed fatal lung conditions as a result of intravenous administration (see Takahashi Y., et al., Cell Transplant. 2011;20(5):727-39). These data convinced them that intralesional application of the stem cells (injections directly into the damaged site of the spinal cord) might be the most effective and reliable method for NS/PC tranplantations.

A new study by the Keio group has attempted to ascertain the efficacy of the intralesional injections. Mice with spinal cord injuries were injected with NS/PCs that had been derived from mice that expression glowing proteins. This allowed the injected cells to be tracked with bio-luminescence imaging (BLI).

The principal investigator of this research is Masaya Nakamura from the Department of Orthopedic Surgery at the Keio University School of Medicine. Dr. Nakamura and his team gave mice spinal contusions at the level of the tenth thoracic vertebra. Then some mice were given low doses and others high doses of NS/PCs that were derived from fetal mice (for those who are interested, low dose – 250,000 cells per mouse; high dose – 1 million cells per mouse) nine days after spinal cord injury. These mice were further divided into two groups: those injected at the lesion epicenter (E), those injected at sites at the front and back of the lesion (RC for “rostral/caudal”). Thus there were four groups total: High dose E, High dose RC, Low dose E, and Low dose RC.

All four groups showed better functional recovery than the control group, which was injected with phosphate buffered saline. BLI showed that the number of cells that survived in each of the four cell-transplanted groups was about the same across these groups.  Thus injecting more cells does not lead to greater numbers of surviving neural stem cells.  This makes sense, since the damaged spinal cord in  very inhospitable place for transplanted cells.

However, when the mice were examined for the expression of particular brain-derived neurotropic factors, the expression of such genes was higher in the RC-injected mice than in the E-injected mice. These results seems to explain why the transplanted NS/PCs differentiated more readily into neurons in the RC-injected mice rather than a type of glial cell known as an astrocyte, as was the case in the E-injected mice.

Human Astrocytes
Human Astrocytes

Nakamura and his team interpreted these results to mean that the environments of the E and RC sites can both support the survival of transplanted NS/PCs during the sub-acute phase of spinal cord injury. The authors conclude with a practical note: “Therefore, we conclude that it is optimal to graft a certain threshold number of NS/PCs into the epicenter lesion during the sub-acute phase of SCI, and thereby avoid causing further iatrogenic injury to the intact RC regions of the spinal cord.”

Hopefully Nakamura’s work will be translated into further human clinical trials. One feature of this study is that a particular threshold of stem cells survive when injected into the spinal cord and injecting larger numbers of cells does not increase the number of surviving cells. Injecting more cells might only contribute to the cell debris in the spinal cord. This is certainly a good thing to know when conducting clinical trials with neural stem cells in spinal cord-injured patients.

Studying Tough-to-Examine Disease by Using Brain Cells Made from Stem Cells

Diseases that are hard to study, such as Alzheimer’s, schizophrenia, and autism can be examined more safely and effectively thanks to an innovative new method for making mature brain cells from reprogrammed skin cells. Gong Chen, the Verne M. William Chair in Life Sciences and professor of biology at Penn State University and the leader of the research team that designed this method said this: “The most exciting part of this research is that it offers the promise of direct disease modeling, allowing for the creation, in a Petri dish, of mature human neurons that behave a lot like neurons that grow naturally in the human brain.”

Chen’s method could lead to customized treatment for individual patients that are based on their own genetic and cellular profile. Chen explained it this way: “Obviously we do not want to remove someone’s brain to experiment on, so recreating the patient’s brain cells in a Petri dish is the next best thing for research purposes and drug screening.”

In previous work, scientists at the University of Wisconsin in James Thomson’s laboratory and in Shinya Yamanaka’s laboratory at Kyoto University in Kyoto, Japan discovered a way to reprogram adult cells into pluripotent stem cells. Such stem cells are called induced pluripotent stem cells or iPSCs. To make iPSCs, scientists infect adult cells with genetically engineered viruses that introduce four specific genes (OCT4, SOX2, KLF4 and cMYC for those who are interested). These genes encode transcription factors, which are proteins that bind to DNA or to the machinery that directly regulates gene expression.  These transcription factors turn on those genes (e.g., OCT4, NANOG, REX1, DNMT3β and SALL4, and OCT4) that induce pluripotency, which means the ability to form any adult cell type.  Once in the pluripotent state, iPSCs can be cultured and grown life embryonic stem cells and can differentiate into adult cell types and tissues.

As Chen explained, “A pluripotent stem cell is a kind of blank slate.”  Chen continued, “During development, such stem cells differentiate into many diverse specialized cell types, such as a muscle cell, a brain cell, or a blood cell.  So, after generating iPSCs from skin cells, researchers then can culture them to become brain cells, or neurons, which can be studies safely in a Petri dish.”

Chen’s team invented a protocol to differentiate iPSCs into mature human neurons much more effectively than previous protocols.  This generates cells that behave neurons in our own brains and can be used to model the individualized disease of a single patient.

In the brain, neurons rarely work alone, but instead are usually in close proximity to star-shaped cells called astrocytes.  Astrocytes are very abundant cells and they assist neuron function and mediate neuronal survival.  “Because neurons are adjacent to astrocytes in the brain, we predicted that this direct physical contact might be an integral part of neuronal growth and health,” said Chen.  To test this hypothesis, Chen and his colleagues began by culturing iPSCs-derived neural stem cells, which are stem cells that have the potential to become neurons.  These cells were cultured on top of a one-cell-thick layer of astrocytes sop that the two cell types were physically touching each other.


“We found that these neural stem cells cultured on astrocytes differentiated into mature neurons much more effectively,” Chen said.  This contrasts Chen’s method with other neural stem cells that were cultured alone in a Petri dish.  As Chen put it, the astrocytes seems to be “cheering the stem cells on, telling them what to do, and helping them to fulfill their destiny to become neurons.”

While this sounds a little cheesy, it is undeniable that the astrocyte layer increases the efficiency of neuronal differentiation of iPSCs.  Personalized medicine is moving beyond the gene level, to the level of cellular organization and tissue physiology, and iPSCs are showing the way.

A Model System for A Devastating Childhood Disease

A Japanese research team from Fukuoka, Japan, specifically from the Department of Pediatrics at the University of Fukuoka, Japan, have used induced pluripotent stem cell technology to make neurons from human patients who suffer from a rare, devastating condition known as Dravet syndrome as a model system.

Dravet syndrome (DS) causes difficult to control seizures within the first year or two of life and later causes cognitive deficits and autistic traits. Dravet’s syndrome is caused by genetic alterations in the SCN1A gene, which encodes the α-subunit of the voltage-gated sodium channel.

DS is very rare – 1/30,000 children, but the mutation is typically not inherited from either parent, but occurs spontaneously in the baby’s cells during development. The best model systems to date are genetically engineered mice, but the differences between human and mouse brains limits the usefulness of this model system.

To make a better model system, workers from the laboratory of Shinichi Hirose took skin biopsy samples from a DS patient, and converted those skin cells into induced pluripotent stem cells (iPSCs), which were then differentiated into neurons. In particular, the neurons that malfunction in DS patients are GABAminergic neurons, and by differentiating DS iPSCs into GABAminergic neurons, Hirose’s laboratory made a model system for DS patients that could be grown in a laboratory culture dish.

Hirose explained their results this way: “From research in mice we believed that SCN1A mutations affect GABAminergic neurons in the forebrain from signaling properly. From the human neurons we also found that GABAminergic neurons were affected by DS, especially during intense stimulation. These patient-specific cell provide an unparalleled insight into the mechanism behind DS and a unique platform for drug development.”

Perhaps such experiments could eventually lead to regenerative treatments for DS patients as well.