Ann M. Parr

TRANSPLANTED ADULT SPINAL CORD-DERIVED NEURAL STEM/PROGENITOR CELLS PROMOTE EARLY FUNCTIONAL RECOVERY AFTER RAT SPINAL CORD INJURY

We examined the effect of spinal cord-derived neural stem/progenitor cells (NSPCs) after delayed transplantation into the injured adult rat spinal cord with or without earlier transplantation of bone marrow-derived mesenchymal stromal cells (BMSCs). Either BMSCs or culture medium were transplanted immediately after clip compression injury (27 g force), and then, 9 days after injury, NSPCs or culture medium were transplanted. Cell survival and differentiation, functional recovery, retrograde axonal tracing, and immunoelectron microscopy were assessed. A significant improvement in functional recovery based on three different measures was seen only in the group receiving NSPCs without BMSCs, and the improved recovery was evident within 1 week of transplantation. In this group, NSPCs differentiated mainly into oligodendrocytes and astrocytes, there was ensheathing of axons at the injury site by transplanted NSPCs, an increase in host oligodendrocytes, and a trend toward an increase in retrogradely labeled supraspinal nuclei. Transplantation of the BMSC scaffold resulted in a trend toward improved survival of the NSPCs, but there was no increase in function. Thus, transplantation of adult rat NSPCs produced significant early functional improvement after spinal cord injury, suggesting an early neuroprotective action associated with oligodendrocyte survival and axonal ensheathment by transplanted NSPCs.

Fate of transplanted adult neural stem/progenitor cells and bone marrow–derived mesenchymal stromal cells in the injured adult rat spinal cord and impact on functional recovery

BACKGROUND Neural stem/progenitor cells derived from the ependymal region of the spinal cord may have the ability to regenerate the injured mammalian spinal cord as they do in some lower vertebrates. It has also been suggested that BMSCs provide an environment conducive to regeneration in the injured cord. METHODS In the current study, both spinal cord-derived NSPCs and BMSCs were cultured from adult male rats expressing eGFP. Neurospheres or dissociated BMSCs were transplanted 9 days after clip compression injury (35-g force). Cell survival and fate, and functional recovery were examined after 14 weeks. RESULTS BMSCs showed no neural differentiation but had much better survival than NSPCs. Transplanted NSPCs differentiated mainly into astrocytes (14.7%) and oligodendrocytes (34.7%), but no neurons. No functional improvement was seen in either transplant group. However, in the NSPC group there was a significant inverse correlation between the functional scores and the number of transplanted astrocytes. A separate experiment to test the effect of cyclosporine on survival and fate of transplanted NSPCs showed that high-dose (20 mg/kg per day) cyclosporine improved cell survival, but had no effect on cell fate. CONCLUSIONS Further work is required before these transplantation strategies can be recommended for patients. These results are promising in that we have found potentially beneficial mechanisms of action of the transplanted cells including differentiation of many NSPCs into oligodendrocytes with the possibility of promoting remyelination, and potential axonal guidance through guiding strands of matrix generated by the BMSCs.

Intrathecal epidermal growth factor and fibroblast growth factor-2 exacerbate meningeal proliferative lesions associated with intrathecal catheters.

OBJECTIVEThere is evidence that promoting the proliferation of endogenous neural stem cells in the spinal cord could be an effective strategy in the treatment of spinal cord injury. We studied this proliferation in a minimal injury rat model in the presence of epidermal growth factor and fibroblast growth factor-2. METHODSAdult female Sprague Dawley rats were minimally injured at T8 and received the growth factors intrathecally by osmotic pump for either 3, 7, or 14 days beginning immediately after spinal cord injury. The infusions were made through a subarachnoid catheter connected to an osmotic minipump. Beginning at the time of injury, the rats received daily bromodeoxyuridine to label proliferating cells for determination of the labeling index. RESULTSSignificant differences were found in the labeling index between injured and non-injured rats. We concluded that minimal spinal cord injury from the stab injury and from the catheter itself increased the proliferation of ependymal region stem/progenitor cells. A minority (23.3%) of the rats developed proliferative lesions in association with the catheters alone, but the lesions were significantly larger and occurred in almost all (93.8%) rats receiving the mitogens, resulting in marked spinal cord compression. CONCLUSIONThe large proliferative growths in the epidermal growth factor and fibroblast growth factor-2-treated rats were unexpected and are indicative of the major effects of these mitogens. This suggests that considerable caution is required in devising therapeutic strategies directed toward mitogenic stimulation by growth factors delivered into the subarachnoid space. Further study is required to determine the appropriate and safe dosage of mitogens for potential use in spinal cord repair.

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.

Emerging Safety of Intramedullary Transplantation of Human Neural Stem Cells in Chronic Cervical and Thoracic Spinal Cord Injury

BACKGROUND Human central nervous system stem cells (HuCNS-SC) are multipotent adult stem cells with successful engraftment, migration, and region-appropriate differentiation after spinal cord injury (SCI). OBJECTIVE To present data on the surgical safety profile and feasibility of multiple intramedullary perilesional injections of HuCNS-SC after SCI. METHODS Intramedullary free-hand (manual) transplantation of HuCNS-SC cells was performed in subjects with thoracic (n = 12) and cervical (n = 17) complete and sensory incomplete chronic traumatic SCI. RESULTS Intramedullary stem cell transplantation needle times in the thoracic cohort (20 M HuCNS-SC) were 19:30 min and total injection time was 42:15 min. The cervical cohort I (n = 6), demonstrated that escalating doses of HuCNS-SC up to 40 M range were well tolerated. In cohort II (40 M, n = 11), the intramedullary stem cell transplantation needle times and total injection time was 26:05 ± 1:08 and 58:14 ± 4:06 min, respectively. In the first year after injection, there were 4 serious adverse events in 4 of the 12 thoracic subjects and 15 serious adverse events in 9 of the 17 cervical patients. No safety concerns were considered related to the cells or the manual intramedullary injection. Cervical magnetic resonance images demonstrated mild increased T2 signal change in 8 of 17 transplanted subjects without motor decrements or emerging neuropathic pain. All T2 signal change resolved by 6 to 12 mo post-transplant. CONCLUSION A total cell dose of 20 M cells via 4 and up to 40 M cells via 8 perilesional intramedullary injections after thoracic and cervical SCI respectively proved safe and feasible using a manual injection technique.

Directed Differentiation of Oligodendrocyte Progenitor Cells From Mouse Induced Pluripotent Stem Cells.

Several neurological disorders, such as multiple sclerosis, the leukodystrophies, and traumatic injury, result in loss of myelin in the central nervous system (CNS). These disorders may benefit from cell-based therapies that prevent further demyelination or are able to restore lost myelin. One potential therapeutic strategy for these disorders is the manufacture of oligodendrocyte progenitor cells (OPCs) by the directed differentiation of pluripotent stem cells, including induced pluripotent stem cells (iPSCs). It has been proposed that OPCs could be transplanted into demyelinated or dysmyelinated regions of the CNS, where they would migrate to the area of injury before terminally differentiating into myelinating oligodendrocytes. OPCs derived from mouse iPSCs are particularly useful for modeling this therapeutic approach and for studying the biology of oligodendrocyte progenitors because of the availability of mouse models of neurological disorders associated with myelin deficiency. Moreover, the utility of miPSC-derived OPCs would be significantly enhanced by the adoption of a consistent, reproducible differentiation protocol that allows OPCs derived from different cell lines to be robustly characterized and compared. Here we describe a standardized, defined protocol that reliably directs the differentiation of miPSCs to generate high yields of OPCs that are capable of maturing into oligodendrocytes.

Assessment of Dysmyelination with RAFFn MRI: Application to Murine MPS I

Type I mucopolysaccharidosis (MPS I) is an autosomal recessive lysosomal storage disorder with neurological features. Humans and laboratory animals with MPS I exhibit various white matter abnormalities involving the corpus callosum and other regions. In this study, we first validated a novel MRI technique, entitled Relaxation Along a Fictitious Field in the rotating frame of rank n (RAFFn), as a measure of myelination and dysmyelination in mice. We then examined differences between MPS I mice and heterozygotes using RAFF5 and histology. RAFF5 (i.e., RAFFn with n = 5) relaxation time constants were highly correlated with histological myelin density (R2 = 0.68, P<0.001), and RAFF5 clearly distinguished between the hypomyelinated and dysmyelinated shiverer mouse and the wild-type mouse. Bloch-McConnell theoretical analysis revealed slower exchange correlation times and smaller exchange-induced relaxation rate constants for RAFF4 and RAFF5 compared to RAFF1-3, T 1ρ, and T 2ρ. These data suggest that RAFF5 may assess methylene protons in myelin lipids and proteins, though other mechanisms (e.g. detection of myelin-bound water) may also explain the sensitivity of RAFF5 to myelin. In MPS I mice, mean RAFF5 relaxation time constants were significantly larger for the striatum (P = 0.004) and internal capsule (P = 0.039), and marginally larger for the fornix (P = 0.15). Histological assessment revealed no differences between MPS I mice and heterozygotes in myelin density or corpus callosum thickness. Taken together, these findings support subtle dysmyelination in the brains of mice with MPS I. Dysmyelination may result from myelin lipid abnormalities caused by the absence of α-L-iduronidase. Our findings may help to explain locomotor and cognitive deficits seen in mice with MPS I.

cGMP-Compliant Expansion of Human iPSC Cultures as Adherent Monolayers.

Therapeutic uses of cells differentiated from human pluripotent stem cells (hPSCs), either embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs), are now being tested in clinical trials, and it is likely that this will lead to increased commercial interest in the clinical translation of promising hPSC research. Recent technical advances in the use of defined media and culture substrates have significantly improved both the simplicity and predictability of growing hPSCs, allowing a much more straightforward application of current good manufacturing practices (cGMP) to the culture of these cells. In addition, the adoption of cGMP-compliant techniques in research environments will both improve the replication of results and make the transition of promising investigations to the commercial sector significantly less cumbersome. However, passaging methods for hPSCs are inherently unpredictable and rely on operator experience and expertise. This is problematic for the cell manufacturing process where operator time and process predictability are often determining cost drivers. We have adopted a human iPSC system using defined media and a recombinant substrate that employs cell dissociation with a hypertonic citrate solution which eliminates variability during hPSC cell expansion and provides a simple cGMP-compliant technique for hiPSC cultivation that is appropriate in both research and commercial applications.

Defined Culture Conditions Accelerate Small-molecule-assisted Neural Induction for the Production of Neural Progenitors from Human-induced Pluripotent Stem Cells:

The use of defined conditions for derivation, maintenance, and differentiation of human-induced pluripotent stem cells (hiPSCs) provides a superior experimental platform to discover culture responses to differentiation cues and elucidate the basic requirements for cell differentiation and fate restriction. Adoption of defined systems for reprogramming, undifferentiated growth, and differentiation of hiPSCs was found to significantly influence early stage differentiation signaling requirements and temporal kinetics for the production of primitive neuroectoderm. The bone morphogenic protein receptor agonist LDN-193189 was found to be necessary and sufficient for neural induction in a monolayer system with landmark antigens paired box 6 and sex-determining region Y-box 1 appearing within 72 h. Preliminary evidence suggests this neuroepithelium was further differentiated to generate ventral spinal neural progenitors that produced electrophysiologically active neurons in vitro, maintaining viability posttransplantation in an immunocompromised host. Our findings support current developments in the field, demonstrating that adoption of defined reagents for the culture and manipulation of pluripotent stem cells is advantages in terms of simplification and acceleration of differentiation protocols, which will be critical for future clinical translation.

Safety and Efficacy of Rose Bengal Derivatives for Glial Scar Ablation in Chronic Spinal Cord Injury

There are no effective therapies available currently to ameliorate loss of function for patients with spinal cord injuries (SCIs). In addition, proposed treatments that demonstrated functional recovery in animal models of acute SCI have failed almost invariably when applied to chronic injury models. Glial scar formation in chronic injury is a likely contributor to limitation on regeneration. We have removed existing scar tissue in chronically contused rat spinal cord using a rose Bengal-based photo ablation approach. In this study, we compared two chemically modified rose bengal derivatives to unmodified rose bengal, both confirming and expanding on our previously published report. Rats were treated with unmodified rose bengal (RB1) or rose bengal modified with hydrocarbon (RB2) or polyethylene glycol (RB3), to determine the effects on scar components and spared tissue post-treatment. Our results showed that RB1 was more efficacious than RB2, while still maintaining minimal collateral effects on spared tissue. RB3 was not taken up by the cells, likely because of its size, and therefore had no effect. Treatment with RB1 also resulted in an increase in serotonin eight days post-treatment in chronically injured spinal cords. Thus, we suggest that unmodified rose Bengal is a potent candidate agent for the development of a therapeutic strategy for scar ablation in chronic SCI.