Researchers at the University of Maryland School of Medicine (UMSOM) have for the first time identified stem cells in the region of the optic nerve, which transmits signals from the eye to the brain.
The finding, published this week in the journal Proceedings of the National Academy of Sciences (PNAS), presents a new theory on why the most common form of glaucoma may develop and provides potential new ways to treat a leading cause of blindness in American adults.
“We believe these cells, called neural progenitor cells, are present in the optic nerve tissue at birth and remain for decades, helping to nourish the nerve fibers that form the optic nerve,” said study leader Steven Bernstein, MD, Ph.D., Professor and Vice Chair of the Department of Ophthalmology and Visual Sciences at the University of Maryland School of Medicine.
“Without these cells, the fibers may lose their resistance to stress, and begin to deteriorate, causing damage to the optic nerve, which may ultimately lead to glaucoma.”
The study was funded by the National Institutes of Health’s National Eye Institute (NEI), and a number of distinguished researchers served as co-authors on the study.
More than 3 million Americans have glaucoma, which results from damage to the optic nerve, causing blindness in 120,000 U.S. patients.
This nerve damage is usually related to increased pressure in the eye due to a buildup of fluid that does not drain properly.
Blind spots can develop in a patient’s visual field that gradually widen over time.
“This is the first time that neural progenitor cells have been discovered in the optic nerve.
Without these cells, the nerve is unable to repair itself from damage caused by glaucoma or other conditions.
This may lead to permanent vision loss and disability,” said Dr. Bernstein.
“The presence of neural stem/progenitor cells opens the door to new treatments to repair damage to the optic nerve, which is very exciting news.”
To make the research discovery, Dr. Bernstein and his team examined a narrow band of tissue called the optic nerve lamina.
Less than 1 millimeter wide, the lamina lies between the light-sensitive retina tissue at the back of the eye and the optic nerve. The long nerve cell fibers extend from the retina through the lamina, into the optic nerve.
What the researchers discovered is that the lamina progenitor cells may be responsible for insulating the fibers immediately after they leave the eye, supporting the connections between nerve cells on the pathway to the brain.
The stem cells in the lamina niche bathes these neuron extensions with growth factors, as well as aiding in the formation of the insulating sheath. The researchers were able to confirm the presence of these stem cells by using antibodies and genetically modified animals that identified the specific protein markers on neuronal stem cells.
“It took 52 trials to successfully grow the lamina progenitor cells in a culture,” said Dr. Bernstein, “so this was a challenging process.”
Dr. Bernstein and his collaborators needed to identify the correct mix of growth factors and other cell culture conditions that would be most conducive for the stem cells to grow and replicate.
Eventually the research team found the stem cells could be coaxed into differentiating into several different types of neural cells.
These include neurons and glial cells, which are known to be important for cell repair and cell replacement in different brain regions.
This discovery may prove to be game-changing for the treatment of eye diseases that affect the optic nerve. Dr. Bernstein and his research team plan to use genetically modified mice to see how the depletion of lamina progenitor cells contributes to diseases such as glaucoma and prevents repair.
Future research is needed to explore the neural progenitors repair mechanisms. “If we can identify the critical growth factors that these cells secrete, they may be potentially useful as a cocktail to slow the progression of glaucoma and other age-related vision disorders.” Dr. Bernstein added.
“This exciting discovery could usher in a sea change in the field of age-related diseases that cause vision loss,” said E. Albert Reece, MD, Ph.D., MBA, Executive Vice President for Medical Affairs, UM Baltimore, and the John Z. and Akiko K. Bowers Distinguished Professor and Dean, University of Maryland School of Medicine. “New treatment options are desperately needed for the millions of patients whose vision is severely impacted by glaucoma, and I think this research will provide new hope for them.”
The primary cause of blindness in humans is glaucoma, an optic neuropathy characterized by progressive degeneration of the optic nerve due to increased intraocular pressure over time. Glaucoma is a progressive disease which can lead to permanent vision loss within a few years in the absence of treatment.
There are two types of glaucoma, open-angle glaucoma and angle-closure glaucoma. The majority of cases in the United States and Western Europe are the former type, while the latter is most common in China and other Asian countries. Both types of glaucoma result from improper drainage of aqueous humor from the eye (Weinreb et al., 2016).
While angle-closure glaucoma results from improper drainage of aqueous humor from the eye (Weinreb et al., 2016) the causes of open-angle glaucoma are now known to be more complex than simple increase in intraocular pressure (Levin, 2005).
The pathophysiology of glaucoma is not well understood, but intraocular pressure is thought to contribute to damage to the optic nerve head with a corresponding loss of retinal ganglion cells.
Other causes of damage in open-angle glaucoma include loss of neurotrophic factors and changes in underlying supporting cells such as glia (Almasieh et al., 2012).
Current treatments for glaucoma focus only on intraocular pressure reduction and this approach is only partly successful.
Alternative and/or supplemental treatments for glaucoma and related optic nerve diseases are needed. The present disclosure is directed to such needs and other important goals.
ONLR Stem Cell Niche Gene Expression
The ONLR contains a neural stem cell niche. To determine whether the ONLR stem cell niche plays a role in the prevention or resistance to glaucoma, gene expression of cells from the niche was examined.
Fresh (< 1 hr old) tissue from young (4-6 year old) male and female rhesus macaque monkeys that previously had no ocular disease or conditions that would alter ocular gene expression was isolated.
This included the eye and attached optic nerve. This tissue was kept on ice until further dissection. The retina, the optic nerve lamina containing the neural stem cell niche (first 0.5cm of the optic nerve, containing the optic nerve head), and distal optic nerve tissues (>lcm behind the laminar region) was dissected.
These tissues were isolated from the original tissue and stored at -80°C until use.
Total RNA was isolated using a sequential purification via RNA Bee (guanidinium thiocyanate-phenol), followed by resuspension in solution provided in the Qiaprep RNA isolation kit.
The resuspended RNA was re-isolated through Qiaprep RNA isolation columns, yielding ultrapure total RNA. Total RNA was used for sequence analysis. Two independent analyses were performed.
First, RNA was utilized from one sample of lamina and one sample of optic nerve. These RNA samples were sent to the Yale sequencing facility for further processing into complementary strand RNA and illumina RNA sequencing and sequence comparison using standard sequence analysis procedures.
Total RNA prepared from a new lamina tissue (the second animal), as well as total RNA from retina from two animals and two optic nerves were sent to the University of Maryland, Baltimore, Biocore for further processing into complementary strand DNA and illumina RNA sequencing.
Thus, there were a total of two retina samples, two lamina samples and two optic nerve samples used for sequencing. Analysis was performed using the Qiagen sequence ingenuity program analysis (IPA) analysis software package. Gene expression was compared by combining CPM sequence results from retina and optic nerve, and related to lamina CPM, using the following formula:
Results from genes associated with growth factors were identified and then grouped together. The growth factors with Lamina/Retina +Optic Nerve ratios >3.0 were further identified. Two groups of genes were thus established: 1) total gene comparison, and 2) growth- factor associated genes that are differentially expressed at higher levels in the lamina stem cell niche.
Genes with laminar niche/surrounding tissue ratios >3.0 and Laminar CPM counts >90 were identified. The chromosomal localization of each gene was confirmed using Genecards (see the website having the URL ending with genecards.org/), and a Google search was performed to determine whether this chromosomal location was reported to be associated with the optic nerve disease glaucoma (either primary open-angle glaucoma, juvenile open-angle glaucoma, normal tension glaucoma, or congenital glaucoma).
The results presented in Table 7 are strong evidence that the ONLR neural stem cell niche expresses genes that are responsible for resistance to developing glaucoma, and also identify the growth factors and related proteins that are involved in this resistance. The products of these genes can be either prepared as the
active peptide, or the whole protein, and administered as a cocktail of the individual factors, such as in the artificial ONLR-NPC extracts of the invention defined herein.
This data demonstrates that the rodent and human postnatal ONLR contains nestin (+)/SOX2(+)/NG2(-) NPCs that give rise to oligodendrocyte progenitor cells (OPCs) and astrocytes. Self-replenishing neural-derived cells that are found within a dedicated vascular plexus give rise to culturable neurospheres, express multiple stem cell marker proteins, differentiate into multiple neural forms, and are age-depleted; these cells that fulfill the criteria of adult NPCs.
ONLR-NPCs are distinct from the nestin (-)/NG2(+) ON-OPCs, which are distinguished by their relative inability to generate neurospheres, grow poorly, and give rise mainly to oligodendrocytes. ONLR-NPCs resemble ‘atypical astrocytes’ previously reported to occur in the ONLR region (Wang et al., 2017).
It is hypothesized that ONLR-NPCs likely perform multiple functions: during the early postnatal period, these cells enable focal myelination in the growing axons, enhancing directional polarization of axonal myelination and also generate astrocytes for the growing ON. ONLR-NPCs may provide a gliogenic processing center during ON growth, contributing to myelination and astrocyte production.
In the mature ON, ONLR-NPCs enhance cellular replacement from stress-related loss and may also supply specific growth factors needed for normal axonal survival and function, similar to that seen in other CNS niches (Lu et al., 2003).
NG2(+) cells comprise 2-8% of the ON (Kang et al., 2010), yet the ON has reduced capacity for remyelination compared with spinal cord (SC) (Lachapelle et al., 2005). If ON remyelination capacity arises from the ONLR-NPC population, then effective repair can occur only in the anterior nerve, while SC-NPC remyelination can occur throughout the spinal cord.
The increased ratio of SOX2(+) nuclei to ONLR total nuclei in 2m/o adult mice, vs the distal ON, is maintained through at least 7 PN months. Supporting these findings, the mouse ONLR formed neurospheres at a much higher frequency compared with distal ON tissue (graph, Fig 2E).
The ONLRs ability to form neurospheres was present in both young and adult animals. It is also present in ONLR from young human donors. The culture data confirms that that SOX2(+) cells in human and rodent ONLR-derived neurospheres co-express nestin(+). These data support the hypothesis that the SOX2(+)/nestin(+) cells present in the mammalian ONLR likely represent a replicating NPC population, endowing the ONLR with a greater response repertoire than that of the distal ON.
Many NG2(+) cells were generated during ONLR-NPC culture, suggesting a preference for NPC > OPC differentiation. Terminal differentiation of cells from ONLR cultures generated both astrocytes and oligodendrocytes, with few (<3%) NeuN (+) or Tuj l(+) cells. This is consistent with the ONLR-NPCs’ hypothesized role in glial replenishment. This was confirmed by ON immunohistochemistry following induction of SOX2-GFP double mutants, which yields GFP(+)/NG2(-) cells in the ONLR, and GFP(+) cells with both astrocytic and oligodendroglial cell morphology in the anterior ON, whereas GFP(+) cells in the distal portion of same nerves exhibit only either NG2(+) or oligodendrocyte morphology. These results support the hypothesis that one role for ONLR-NPCs is to enable in vivo generation of mature glia.
Postnatal axonal growth can occur at specific stress points located at myelin: unmyelinated junctions (Goldberg, 2003). Since the region adjacent to the ONLR is where RGC axonal myelination begins, these results suggest that postnatal ON growth occurs by axonal extension from the unmyelinated retina, with myelination directly associated with emergence from the ONLR.
ONLR-NPCs provide a source of oligodendrocytes and astrocytes during intensive postnatal ON growth. Without adequate ONLR-NPCs, axons either remain hypomyelinated or die. The data herein show both axonal loss and anterior hypomyelination following selective depletion of ONLR-NPCs following exposure to 4-OHT in SOX2-DTA reporter mice. In contrast, more distal axonal segments had a nearly normal myelination pattern.
In adults, overall oligodendrocyte generation declines, but the ONLR continues to have an increased Ki67(+) mitotic index, suggesting that the ONLR niche continues to contribute to adult glial cell replacement. It is hypothesized that ONLR-NPCs have distinct roles during early postnatal and adult stages: in the young, ONLR-NPCs contribute to ON growth, while in the adult they contribute to glial cell replacement.
Mouse ONLR-SOX2(+) nuclear numbers decline during aging, but remain relatively
constant in the distal ON. An age-related decline in human ONLR-nestin expression is demonstrable (see Fig 5i). These data indicate age-dependent ONLR-NPC loss. Human ONLR- NPCs are ultimately lost during aging, and this loss may alter the balance between degeneration, repair and the emergence of age-related ON diseases.
An important age-associated ON disease is open-angle glaucoma (OAG) (Lee et al., 2014), a progressive neuropathy associated with the ONLR. Progressive resistance to glaucoma treatment occurs in elderly individuals (Rossetti et al., 2010). ONLR-NPC depletion may contribute to OAG progression by reducing gliogenic support and associated neurotrophic factors such as brain-derived growth factor (BDNF), important for retinal ganglion cell (RGC)
survival under stress, and which is secreted by cultured lamina-derived cells (Lambert et al., 2004). It is hypothesized that ONLR-NPCs present in younger individuals may protect against disease progression, and the age-related loss of ONLR-NPCs may contribute to disease progression via the inability to repair stress-related damage.
In summary, the ONLR in both humans and mice is not a non-proliferative barrier, but rather contains an NPC niche, which may have a role in both postnatal ON development and in adult ON support and repair. Age-related ONLR-NPC loss likely contributes to ON diseases such as OAG and understanding the roles of ONLR-NPCs enables new therapeutic strategies to treat ON disease.
While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.
More information: S. L. Bernstein et al, The optic nerve lamina region is a neural progenitor cell niche, Proceedings of the National Academy of Sciences (2020). DOI: 10.1073/pnas.2001858117
