Ellen Bible

The support of neural stem cells transplanted into stroke-induced brain cavities by PLGA particles

Stroke causes extensive cellular loss that leads to a disintegration of the afflicted brain tissue. Although transplanted neural stem cells can recover some of the function lost after stroke, recovery is incomplete and restoration of lost tissue is minimal. The challenge therefore is to provide transplanted cells with matrix support in order to optimise their ability to engraft the damaged tissue. We here demonstrate that plasma polymerised allylamine (ppAAm)-treated poly(D,L-lactic acid-co-glycolic acid) (PLGA) scaffold particles can act as a structural support for neural stem cells injected directly through a needle into the lesion cavity using magnetic resonance imaging-derived co-ordinates. Upon implantation, the neuro-scaffolds integrate efficiently within host tissue forming a primitive neural tissue. These neuro-scaffolds could therefore be a more advanced method to enhance brain repair. This study provides a substantial step in the technology development required for the translation of this approach.

Non-invasive imaging of transplanted human neural stem cells and ECM scaffold remodeling in the stroke-damaged rat brain by 19F- and diffusion-MRI

Transplantation of human neural stem cells (hNSCs) is emerging as a viable treatment for stroke related brain injury. However, intraparenchymal grafts do not regenerate lost tissue, but rather integrate into the host parenchyma without significantly affecting the lesion cavity. Providing a structural support for the delivered cells appears important for cell based therapeutic approaches. The non-invasive monitoring of therapeutic methods would provide valuable information regarding therapeutic strategies but remains a challenge. Labeling transplanted cells with metal-based (1)H-magnetic resonance imaging (MRI) contrast agents affects the visualization of the lesion cavity. Herein, we demonstrate that a (19)F-MRI contrast agent can adequately monitor the distribution of transplanted cells, whilst allowing an evaluation of the lesion cavity and the formation of new tissue on (1)H-MRI scans. Twenty percent of cells labeled with the (19)F agent were of host origin, potentially reflecting the re-uptake of label from dead transplanted cells. Both T(2)- and diffusion-weighted MRI scans indicated that transplantation of hNSCs suspended in a gel form of a xenogeneic extracellular matrix (ECM) bioscaffold resulted in uniformly distributed cells throughout the lesion cavity. However, diffusion MRI indicated that the injected materials did not yet establish diffusion barriers (i.e. cellular network, fiber tracts) normally found within striatal tissue. The ECM bioscaffold therefore provides an important support to hNSCs for the creation of de novo tissue and multi-nuclei MRI represents an adept method for the visualization of some aspects of this process. However, significant developments of both the transplantation paradigm, as well as regenerative imaging, are required to successfully create new tissue in the lesion cavity and to monitor this process non-invasively.

Neo-vascularization of the stroke cavity by implantation of human neural stem cells on VEGF-releasing PLGA microparticles

Replacing the tissue lost after a stroke potentially provides a new neural substrate to promote recovery. However, significant neurobiological and biotechnological challenges need to be overcome to make this possibility into a reality. Human neural stem cells (hNSCs) can differentiate into mature brain cells, but require a structural support that retains them within the cavity and affords the formation of a de novo tissue. Nevertheless, in our previous work, even after a week, this primitive tissue is void of a vasculature that could sustain its long-term viability. Therefore, tissue engineering strategies are required to develop a vasculature. Vascular endothelial growth factor (VEGF) is known to promote the proliferation and migration of endothelial cells during angio- and arteriogenesis. VEGF by itself here did not affect viability or differentiation of hNSCs, whereas growing cells on poly(D,L-lactic acid-co-glycolic acid) (PLGA) microparticles, with or without VEGF, doubled astrocytic and neuronal differentiation. Secretion of a burst and a sustained delivery of VEGF from the microparticles in vivo attracted endothelial cells from the host into this primitive tissue and in parts established a neovasculature, whereas in other parts endothelial cells were merely interspersed with hNSCs. There was also evidence of a hypervascularization indicating that further work will be required to establish an adequate level of vascularization. It is therefore possible to develop a putative neovasculature within de novo tissue that is forming inside a tissue cavity caused by a stroke.

Attachment of stem cells to scaffold particles for intra-cerebral transplantation

Cell-replacement therapy and tissue regeneration using stem cells are of great interest to recover histological damage caused by neuro-degenerative disease or traumatic insults to the brain. To date, the main intra-cerebral delivery for these cells has been as a suspension in media through a thin needle. However, this does not provide cells with a support system that would allow tissue regeneration. Scaffold particles are needed to provide structural support to cells to form de novo tissue. In this 16-d protocol, we describe the generation and functionalization of poly (D,L-lactic-co-glycolic) acid (PLGA) particles to enhance cell attachment, the attachment procedure to avoid clumping and aggregation of cells and particles, and their preparation for intra-cerebral injection through a thin needle. Although the stem cell–scaffold transplantation is more complicated and labor-intensive than cell suspensions, it affords de novo tissue generation inside the brain and hence provides a significant step forward in traumatic brain repair.

Distinct patterns of serum immunoreactivity as evidence for multiple brain-directed autoantibodies in juvenile neuronal ceroid lipofuscinosis.

Autoantibodies to glutamic acid decarboxylase (GAD65) have been reported in sera from the Cln3–/– mouse model of juvenile neuronal ceroid lipofuscinosis (JNCL), and in individuals with this fatal paediatric neurodegenerative disorder. To investigate the existence of other circulating autoreactive antibodies, we used sera from patients with JNCL and other forms of neuronal ceroid lipofuscinosis (NCL) as primary antisera to stain rat and human central nervous system sections. JNCL sera displayed characteristic patterns of IgG, but not IgA, IgE or IgM immunoreactivity that was distinct from the other forms of NCL. Immunoreactivity of JNCL sera was not confined to GAD65‐positive (GABAergic) neurons, but also stained multiple other cell populations. Preadsorption of JNCL sera with recombinant GAD65 reduced the intensity of the immunoreactivity, but did not significantly change its staining pattern. Moreover, sera from Stiff Person Syndrome and Type I Diabetes, disorders in which GAD65 autoantibodies are present, stained with profiles that were markedly different from JNCL sera. Collectively, these studies provide evidence of the presence of autoreactive antibodies within multiple forms of NCL, and are not exclusively directed towards GAD65.

AAV-mediated gene therapy causes functional improvements in a murine model of infantile neuronal ceroid lipofuscinosis (INCL, Batten disease)

Batten Disease (Neuronal Ceroid Lipofuscinoses, NCL) refers to a group of inherited neurodegenerative disorders that primarily affect children. Affected children have a disease progression that consists of blindness, seizures, motor and metal decline, and finally premature death. The form with the earliest onset is Infantile Neuronal Ceroid Lipofuscinosis (INCL), and is caused by mutations in the gene that codes for the lysosomal hydrolase palmitoyl protein thioesterase-1 (PPT1). A murine model of INCL, that lacks PPT1 activity, displays many of the same phenotypic characteristics of children with INCL, including: visual deficits, spontaneous seizures, motor deficits, and reduced longevity. To determine if an AAV-mediated gene therapy approach, using an AAV2 vector containing the human PPT1 cDNA (AAV-PPT1), could produce functional improvements in the murine model INCL, we treated newborn PPT1-deficient mice with four intracranial (I.C.) injections of AAV-PPT1 (0.5 ul/site, 1×E10 IU/ml). This group was examined for seizure phenotype, motor function, and longevity of the treated mice.