A representative confocal image of spinal cord tissue fluorescently immunolabeled for SC121 (red) in conjunction with GFAP (green) – markers that allowed researchers to quantify stem cell differentiation and migration. (Image provided by study author Dr. Aileen J. Anderson)

Research has shown that transplanting human neural stem cells into damaged spinal cords restores locomotor function in a mouse model of spinal cord injury¹. Researchers who worked on that study have published another paper examining how these neural stem cells behave in injured tissue as they aid in healing. Learning how stem cells behave in injured tissue will hopefully help researchers develop better treatments for spinal cord injuries.

In the study, researchers used Stereo Investigator to stereologically quantify the survival, migration, proliferation, and differentiation of human neural stem cells transplanted into injured and uninjured mice. Stem cells were analyzed in mouse brain tissue specimens 1, 7, 14, 28, and 98 days after transplantation. The research found that there were fewer stem cells in the injured animals compared to the uninjured animals at all time points, stem cells in injured mice localized near the center of the injury, a delay of stem cell proliferation in injured tissue led to an overall deficit of actively dividing cells, proliferation in injured mice occurred closer to the injection sites (the locations where the stem cells were injected into the mice), and the injured microenvironment increased differentiation to more mature oligodendrocytes.

SC121, a marker that binds to human cytoplasm in cells, was used to identify human neural stem cells in the tissue sections of mouse spinal cord. The researchers stereologically quantified this marker to find the number of human neural stem cells in the injured and uninjured tissue at each time point. Analysis at one day post-transplantation showed that only 11% of the initial transplant dose survived in injured animals while 25% survived in uninjured animals. The number of stem cells in each group increased over time, however, there were fewer stem cells in the injured animals compared to the uninjured animals at all time points.

To analyze the migration of stem cells, researchers used Stereo Investigator to quantify the number of stem cells at varying distances from the injection sites (the locations where the stem cells were injected into the mice), and expressed these localized counts as percentages of the total number of cells counted. At 98 days post transplantation there was a significant difference in the distribution of stem cells between injured and uninjured spinal cords; stem cells in injured mice localized near the center of the injury.

Proliferation was quantified by using Stereo Investigator to count the number of cells that express SC121 and BrdU or KI67 – an indication that cells are in the process of dividing. Very few cells were actively dividing 1 day post transplantation and there was no significant increase in the number of actively dividing cells in injured tissue specimens, except when comparing 28 days post transplantation data to 1 day post transplantation. This delay of stem cell proliferation in the first 28 days in injured tissue led to an overall deficit of actively dividing cells throughout the entire period. Also interesting is that with all of the cell division activity, there were no tumors detected.

Then, the researchers wanted to see where proliferation was occurring and where the stem cells migrated to. They quantified this by taking the numbers of cells expressing SC121 and BrdU at given distances from the site of injection and expressing them as percentages of the total number of cells counted. Proliferation in injured mice occurred closer to injection site and prior to migration toward the injury epicenter, while stem cells in the uninjured spinal cord proliferated in multiple waves alternating with migration along the length of the spinal cord.

To find out what type of cells the adult stem cells differentiated into, the researchers stereologically quantified stem cells that expressed markers for oligodendrocytic (OLIG2), astrocytic (SC123) and neuronal (DCX) lineages at 98 days post transplantation. They found that the injured tissue had more oligodendritic cells than the uninjured tissue and overall the majority of stem cells differentiated along oligodendrocytic lineage.

Migration and distribution of the differentiated cells were examined at 98 days post transplantation. The researchers found that stem cells along the oligodendrocytic lineage were in localized peaks around the injury epicenter, as opposed to an even distribution along the length of the spinal cord as found in the uninjured tissue. There also was a significant increase in the percentage of SC123 stem cells in injured animals at the center of the injury that was directly proportional to the decrease of OLIG2 cells. This suggests that astrocytes were recruited to the injury site at the expense of oligodendrocytes.

The researchers noticed that many of the stem cells did not have a marker to indicate what type of differentiated cell it is. Since OLIG2 is a marker for precursor/progenitor oligodendrocytes, researchers postulated that these unmarked cells could be older oligodendrocytes. So they used APC/CC1 – a marker for more mature oligodendrocytes. When they did this, the percentage of cells accounted for increased from 71.9% to 97.4%. This suggests that, on the whole, the injured microenvironment increased differentiation to more mature oligodendrocytes.

Stem cell treatments have emerged as an option to help repair nerve damage suffered from spinal cord injuries, however there is still much to be learned about stem cells and how to effectively use them to treat spinal cord injuries. The stereological data and analyses reported in this research study uncover interesting information about how human neural stem cells behave in a mouse model of spinal cord injury: fewer stem cells survived in injured tissue and proliferation was delayed – causing a deficit in the number of stem cells through the length of study. Stem cells proliferated near the injection site and then migrated toward the injury, and they differentiated into more mature oligodendrocytes. Hopefully, the information presented in this study can be used to help find more effective treatments for spinal cord injuries.

¹Cummings, B.J., Uchida, N., Tamaki, S.J., Salazar, D.L., Hooshmand,M., Summers, R., Gage, F.H., and Anderson, A.J. (2005). Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc. Natl. Acad. Sci. USA 102,14069–14074.

Sontag, Christopher J., Uchida, N., Cummings, Brian J., & Anderson, Aileen J. (2014). Injury to the Spinal Cord Niche Alters the Engraftment Dynamics of Human Neural Stem Cells. Stem Cell Reports(0).

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The study, published last month in Nature Biotechnology, “is the first to show that human stem cells can successfully implant themselves in the brain and then heal neurological deficits,” senior author Su-Chun Zhang told the University of Wisconsin-Madison news department.

Using chemicals known to promote neuron development, Dr. Zhang‘s team cultured the stem cells in vitro. Once the cells reached a state of partial differentiation, meaning they were on their way to developing into neurons, they were injected into the hippocampi of a group of mice. The mice, members of a strain that does not reject transplants from other species, had received injury to their medial septum brain region. According to the paper, this brain region is involved in learning and memory, and is connected to the hippocampus by GABA and cholinergic neurons.

The researchers used a variety of methods, including stereology with an MBF system available at the The Waisman Center’s Core Imaging facility (Stereo Investigator software coupled with a Zeiss fluorescence microscope), to analyze the postmortem brains. They determined that the stem cells had successfully developed into basal forebrain cholinergic neurons (BFCNs) and GABAergic neurons that became integrated into the circuitry of the mouse brains.

Behavioral tests such as the Morris water maze, showed that the mice performed better in tasks related to learning and memory after cell transplant, leading the researchers to conclude that the new cells had corrected deficits in these areas.

“Our findings support the prospect of using human stem cell-derived MGE-like progenitors in developing therapies for neurological disorders of learning and memory,” the authors say in their paper.

Liu, Y., Weick, J., Liu, H., Krencik, R., Zhang, X., Ma, L., Zhou, G., Ayala, M., & Zhang, S. (2013). Medial ganglionic eminence–like cells derived from human embryonic stem cells correct learning and memory deficits Nature Biotechnology DOI: 10.1038/nbt.2565

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A stroke can leave its victim mentally and physically devastated. Recovery is demanding, and takes drive and determination. If the patient doesn’t receive medical attention within the small, critical window of time after the stroke occurs, chances of a quick recovery are slim. Developments in stem cell research find doctors optimistic about new possibilities for stroke rehabilitation.

Scientists at the Stanford Stroke Center, one of the leading centers for stroke research in the world, are working on figuring out how transplanted stem cells affect the host brain. They’re specifically working toward determining which trophic factors—substances secreted by cells—are necessary for stem cell transplantation to be effective. One recent study focused on vascular endothelial growth factor (VEGF), a factor associated with neurological recovery in stroke patients.

As described in their paper “Transplanted Stem Cell-secreted VEGF Effects Post-stroke Recovery, Inflammation, and Vascular Repair,” Drs. Nobutaka Horie, Gary Steinberg, and their team of researchers found VEGF to be essential for hCNS-SCns-induced recovery. The research team studied the brains of rats, which had undergone stroke surgery and were injected with human central nervous system stem cells (hCNS-SCns). After thorough analysis of various parameters, including stereological quantification of the brain inflammatory response and stem cell survival using Stereo Investigator, the Stanford team determined the stem cells helped suppress inflammation, helped form new blood vessels, and improved blood-brain barrier integrity.”

“Subacute cell transplantation therapy offers a multimodal strategy for brain repair that could significantly expand the therapeutic window for stroke,” say the authors in “Transplanted Stem Cell-secreted VEGF Effects Post-stroke Recovery, Inflammation, and Vascular Repair.”

Access the free abstract or download the full article at Stem Cells.

Horie, N., Pereira, M. P., Niizuma, K., Sun, G., Keren-Gill, H., Encarnacion, A., Shamloo, M., Hamilton, S. A., Jiang, K., Huhn, S., Palmer, T. D., Bliss, T. M. and Steinberg, G. K. (2011), Transplanted Stem Cell-Secreted Vascular Endothelial Growth Factor Effects Poststroke Recovery, Inflammation, and Vascular Repair. STEM CELLS, 29: 274–285. doi: 10.1002/stem.584

{Image of transplanted stem cells (pink) and blood vessels (green) courtesy of Stanford University}

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Thousands of people in the United States have spinal cord injuries (SCIs), with associated loss of movement and sensation below the site of the injury. Neural and glial cell transplants into research animals after SCI have correlated with recovery of function. The improvement may be caused by the transplanted cells; it’s thought that remyelination by the transplanted glial cells is the main reason for the improvement. Also, if adult neural stem cells are transplanted, there is evidence they form new neurons. In “Analysis of Host-Mediated Repair Mechanisms after Human CNS-Stem Cell Transplantation for Spinal Cord Injury: Correlation of Engraftment with Recovery” (2009, Hooshmand MJ, Sontag CJ, Uchida N, Tamaki S, Anderson AJ, Cummings BJ, PLoS One) the authors use Stereo Investigator’s powerful quantitative tools to determine whether changes in the host environment may also be correlated with improved function.

Using Stereo Investigator, specific aspects of the host milieu were compared between spinal cord injured animals (Non-Obese-
Diabetic-severe combined immunodeficient mice) that received transplants of human central nervous system-stem cell neurospheres (hCNS-SCns) and those that did not. The sampling parameters such as section interval, grid size, and counting frame size, were determined by checking the coefficient of error to make sure it was low. In some cases, additional post-hoc power analysis of data from previous publications was used to demonstrate that the parameters were appropriate for the required precision. Serotonergic fiber length was estimated using the Isotropic Virtual Planes probe with a 60X objective. Blood vessel length was estimated using the Space Balls probe with a 40X objective. The areas and volumes of lesions, spared tissue, and astrogliosis, were estimated using the Cavalieri probe with a 20X objective. Stereological results were complimented by biochemical protein analysis. In addition, the Optical Fractionator probe was used to estimate a non-host parameter, the number of neurons that live and proliferate from the hCNS-SCns transplant.

There were no differences found in the host characteristics between hCNS-SCns transplant animals and control animals. For example, there was no difference in the length of blood vessels. Platelet/ endothelial cell adhesion molecule immunohistochemistry was used to identify blood vessels. Some treatments following CNS trauma may promote behavioral recovery associated with vascular remodeling. Blood vessel length was estimated at the injury center, one mm rostral, and one mm caudal to the injury. There was no statistical difference between controls and hCNS-SCns transplanted animals (see figure, controls are vehicle and human fibroblasts (hFb)). Regarding the non-host characteristic of how many transplanted cells lived, multiplied, and migrated, the Optical Fractionator estimate showed that the transplanted cell number increased 194 percent after transplantation and migrated from the injection site. Ablation of some transplanted cells with Diptheria toxin correlated with a loss of locomotor recovery. This study shows that the direct consequences of the transplanted cells such as proliferation -
correlate with improved function – while the transplant does not have an effect on host characteristics such as lesion volume,
spared tissue, fiber sprouting, and angiogenesis, ruling out any correlation of an indirect effect of the transplanted stem cells with recovery.

Dan Peruzzi is a staff scientist at MBF Bioscience.

First published in The Scope, fall 2009.

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