Henry Klassen

Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord.

Lesion‐induced inflammatory responses in both brain and spinal cord have recently become a topic of active investigation. Using C57BL/6J mice, we compared the tissue reaction in these two central nervous system (CNS) compartments with mechanical lesions of similar size involving both grey and white matter. This evaluation included the quantitative assessment of neutrophils, lymphocytes and activated macrophages/microglia, as well as astrocyte activation, upregulation of vascular cell adhesion molecules (ICAM‐1, VCAM‐1, PECAM) and the extent of blood–brain barrier (BBB) breakdown. Time points analysed post‐lesioning included 1, 2, 4 and 7 days (as well as 10 and 14 days for the BBB). We found clear evidence that the acute inflammatory response to traumatic injury is significantly greater in the spinal cord than in the cerebral cortex. The numbers of both neutrophils and macrophages recruited to the lesion site were significantly higher in the spinal cord than in the brain, and the recruitment of these cells into the surrounding parenchyma was also more widespread in the cord. The area of BBB breakdown was substantially larger in the spinal cord and vascular damage persisted for a longer period. In the brain, as in spinal cord, the area to which neutrophils were recruited correlated well with the area of BBB breakdown. It will be of interest to determine the extent to which the infiltration of inflammatory cells contributes, either directly or indirectly, to the vascular permeability and secondary tissue damage or, conversely, to local tissue repair in the brain and the spinal cord.

Neuronal Differentiation and Morphological Integration of Hippocampal Progenitor Cells Transplanted to the Retina of Immature and Mature Dystrophic Rats

Attempts to repopulate the retina with grafted neurons have been unsuccessful, in large part because donor cells prefer not to integrate with those of the host. Here we describe the first use of neural progenitor cells in the diseased adult retina. Adult rat hippocampal progenitor cells were injected into the eyes of rats with a genetic retinal degeneration. After survival times up to 16 weeks, the retinae of 1-, 4-, and 10-week-old recipients exhibited widespread incorporation of green fluorescent protein-expressing (GFP+) donor cells into the host retina. The 18-week-old recipients showed a similar pattern, but with fewer cells. Grafted cells expressed the mature neuronal markers NF-200, MAP-5, and calbindin. GFP+ cells extended numerous neurites into the host plexiform layers and these processes were intimately associated with synaptophysin+ profiles. GFP+ neurites also extended into the host optic nerve head. These results demonstrate the differentiation of substantial numbers of new neurons within the mature dystrophic retina.

Isolation and characterization of neural progenitor cells from post-mortem human cortex

Post‐mortem human brain tissue represents a vast potential source of neural progenitor cells for use in basic research as well as therapeutic applications. Here we describe five human neural progenitor cell cultures derived from cortical tissue harvested from premature infants. Time‐lapse videomicrography of the passaged cultures revealed them to be highly dynamic, with high motility and extensive, evanescent intercellular contacts. Karyotyping revealed normal chromosomal complements. Prior to differentiation, most of the cells were nestin, Sox2, vimentin, and/or GFAP positive, and a subpopulation was doublecortin positive. Multilineage potential of these cells was demonstrated after differentiation, with some subpopulations of cells expressing the neuronal markers β‐tubulin, MAP2ab, NeuN, FMRP, and Tau and others expressing the oligodendroglial marker O1. Still other cells expressed the classic glial marker glial fibrillary acidic protein (GFAP). RT‐PCR confirmed nestin, SOX2, GFAP, and doublecortin expression and also showed epidermal growth factor receptor and nucleostemin expression during the expansion phase. Flow cytometry showed high levels of the neural stem cell markers CD133, CD44, CD81, CD184, CD90, and CD29. CD133 markedly decreased in high‐passage, lineage‐restricted cultures. Electrophysiological analysis after differentiation demonstrated that the majority of cells with neuronal morphology expressed voltage‐gated sodium and potassium currents. These data suggest that post‐mortem human brain tissue is an important source of neural progenitor cells that will be useful for analysis of neural differentiation and for transplantation studies.

Transplantation of Adult Mouse iPS Cell-Derived Photoreceptor Precursors Restores Retinal Structure and Function in Degenerative Mice

This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs), could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells.

Retinal progenitor cells (RPCs) are multipotent central nervous system precursors that give rise to all of the cell types of the retina during development. Several groups have reported that mammalian RPCs can be isolated and expanded in culture and can differentiate into retinal neurons upon grafting to the mature, diseased eye. However, cell delivery and survival remain formidable obstacles to application of RPCs in a clinical setting. Because biodegradable polymer/progenitor constructs have been shown to be capable of tissue generation in other compartments, we evaluated the survival, migration, and differentiation of RPCs delivered on PLLA/PLGA polymer substrates to the mouse subretinal space and compared these results to conventional injections of RPCs. Polymer composite grafts resulted in a near 10‐fold increase in the number of surviving cells after 4 weeks, with a 16‐fold increase in cell delivery. Grafted RPCs migrated into the host retina and expressed the mature markers neurofilament‐200, glial fibrillary acidic protein, protein kinase C‐α, recoverin, and rhodopsin. We conclude that biodegradable polymer/progenitor cell composite grafts provide an effective means of increasing progenitor cell survival and overall yield when transplanting to sites within the central nervous system such as the retina.

Stem cells and retinal repair.

Retinal stem cells (RSCs) are multipotent central nervous system (CNS) precursors that give rise to the retina during the course of development. RSCs are present in the embryonic eyecup of all vertebrate species and remain active in lower vertebrates throughout life. Mammals, however, exhibit little RSC activity in adulthood and thus little capacity for retinal growth or regeneration. Because CNS precursors can now be isolated from immature and mature mammals and expanded ex vivo, it is possible to study these cells in culture as well as following transplantation to the diseased retina. Such experiments have revealed a wealth of unanticipated findings, both in terms of the instructive cues present in the mature mammalian retina as well as the ability of grafted CNS precursors to respond to them. This review examines current knowledge regarding RSCs, together with other CNS precursors, from the perspective of investigators who wish to isolate, propagate, genetically modify, and transplant these cells as a regenerative strategy with application to retinal disease.

Neural Progenitor Cells Lack Immunogenicity and Resist Destruction as Allografts

Multipotent, self‐renewing stem and progenitor cells isolated from the mammalian central nervous system (CNS) have been shown to survive as allografts following transplantation to sites throughout the neuraxis. However, studies of this type shed little light upon the immunologic properties of the cells themselves, primarily because little is learned about the intrinsic immunogenic properties of a cell when it is grafted into an immune‐privileged site. We have therefore investigated the immunogenic and antigenic properties of CNS progenitor cells by grafting them into a conventional (i.e., non‐immune‐privileged) site, namely, beneath the kidney capsule. Our results indicate that allogeneic CNS progenitor cells survive at least 4 weeks in a conventional site, during which time they neither sensitize their hosts nor express detectable levels of major histocompatibility complex (MHC) class I or II. These in vivo data are in accord with flow cytometric results showing that CNS progenitor cells do not express MHC class I or class II, either at baseline or upon differentiation in 10% serum. Exposure to interferon gamma, however, reversibly upregulates expression of these key transplantation antigens. Together, these results reveal CNS progenitor cells to possess inherent immune privilege. Since CNS progenitor cell allografts were rejected beneath the kidney capsule following specific sensitization of the host, CNS progenitor cells were able to display alloantigens, albeit not in an immunogenic form.

Engineering retinal progenitor cell and scrollable poly(glycerol-sebacate) composites for expansion and subretinal transplantation.

Retinal degenerations cause permanent visual loss and affect millions world-wide. Presently, a novel treatment highlights the potential of using biodegradable polymer scaffolds to induce differentiation and deliver retinal progenitor cells for cell replacement therapy. In this study, we engineered and analyzed a micro-fabricated polymer, poly(glycerol sebacate) (PGS) scaffold, whose useful properties include biocompatibility, elasticity, porosity, and a microtopology conducive to mouse retinal progenitor cell (mRPC) differentiation. In vitro proliferation assays revealed that PGS held up to 86,610 (+/-9993) mRPCs per square millimeter, which were retained through simulated transplantations. mRPCs adherent to PGS differentiated toward mature phenotypes as evidenced by changes in mRNA, protein levels, and enhanced sensitivity to glutamate. Transplanted composites demonstrated long-term mRPC survival and migrated cells exhibited mature marker expression in host retina. These results suggest that combining mRPCs with PGS scaffolds for subretinal transplantation is a practical strategy for advancing retinal tissue engineering as a restorative therapy.

CNS Progenitor Cells Promote a Permissive Environment for Neurite Outgrowth via a Matrix Metalloproteinase-2-Dependent Mechanism

Transplantation of progenitor cells to the CNS has shown promise in neuronal and glial replacement and as a means of rescuing host neurons from apoptosis. Here we examined the effect of progenitor grafts on neurite extension in the degenerating retina of rd1 (retinal degeneration 1) mice. Transplantation of retinal progenitor cells induced increased matrix metalloproteinase-2 (MMP2) secretion, partly from activated glial cells, which was then activated by neuronally expressed MMP14. Active MMP2 resulted in proteolysis of the neurite outgrowth inhibitors CD44 and neurocan in the degenerative retina, allowing significantly increased neurite outgrowth across the border between abutting nondystrophic and rd1 retinas. Progenitor-induced enhancement of outgrowth was abrogated by an MMP inhibitor or by coculture with retinal explants from MMP2−/− mice. This study provides the first identification of an MMP2-dependent mechanism by which exogenous progenitor cells alter the host environment to promote neural regeneration. This suggests a novel therapeutic role for progenitor cells in the treatment of CNS degenerative diseases.

Surface markers expressed by multipotent human and mouse neural progenitor cells include tetraspanins and non-protein epitopes

Surface molecules play important roles in a wide range of cellular functions, yet little has been reported regarding the expression of such markers by neural stem cells. Here, multipotent human neural progenitor cells (hNPCs) were expanded as a monolayer in the presence of fibroblast/epidermal growth factor, harvested, labeled with monoclonal antibodies, and analyzed by flow cytometry. Positive markers included CD9, CD15, CD81, CD95 (Fas), GD(2) ganglioside, and major histocompatibility complex class I and beta2 microglobulin, as well as low levels of the hematopoietic stem cell marker CD34. Of these, mouse NPCs were positive for CD9, CD15, CD81, and GD(2) ganglioside. The markers reported here have been implicated in a wide range of cellular functions including proliferation, migration, differentiation, apoptosis, and immune recognition.