Dustin R. Wakeman

Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease

Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinsons disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinsons disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinsons disease.Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson’s disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson’s disease.

Behavioral improvement in a primate Parkinson's model is associated with multiple homeostatic effects of human neural stem cells

Stem cells have been widely assumed to be capable of replacing lost or damaged cells in a number of diseases, including Parkinsons disease (PD), in which neurons of the substantia nigra (SN) die and fail to provide the neurotransmitter, dopamine (DA), to the striatum. We report that undifferentiated human neural stem cells (hNSCs) implanted into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated Parkinsonian primates survived, migrated, and had a functional impact as assessed quantitatively by behavioral improvement in this DA-deficit model, in which Parkinsonian signs directly correlate to reduced DA levels. A small number of hNSC progeny differentiated into tyrosine hydroxylase (TH) and/or dopamine transporter (DAT) immunopositive cells, suggesting that the microenvironment within and around the lesioned adult host SN still permits development of a DA phenotype by responsive progenitor cells. A much larger number of hNSC-derived cells that did not express neuronal or DA markers was found arrayed along the persisting nigrostriatal path, juxtaposed with host cells. These hNSCs, which express DA-protective factors, were therefore well positioned to influence host TH+ cells and mediate other homeostatic adjustments, as reflected in a return to baseline endogenous neuronal number-to-size ratios, preservation of extant host nigrostriatal circuitry, and a normalizing effect on α-synuclein aggregation. We propose that multiple modes of reciprocal interaction between exogenous hNSCs and the pathological host milieu underlie the functional improvement observed in this model of PD.

Adhesive interactions between human neural stem cells and inflamed human vascular endothelium are mediated by integrins.

Understanding the mechanisms by which stem cells home precisely to regions of injury or degeneration is of importance to both basic and applied regenerative medicine. Optimizing regenerative processes may depend on identifying the range of molecules that subserve stem cell trafficking. The “rolling” of extravasating cells on endothelium under conditions of physiological flow is the first essential step in the homing cascade and determines cell adhesion and transmigration. Using a laminar flow chamber to simulate physiological shear stress, we explored an aspect of this process by using human neural stem cells (hNSCs). We observed that the interactions between hNSCs and tumor necrosis factor‐α (TNF‐α)‐stimulated human endothelium (simulating an inflamed milieu) are mediated by a subclass of integrins—α2, α6, and β1, but not α4, αv, or the chemokine‐mediated pathway CXCR4‐stromal cell‐derived factor‐1α—suggesting not only that the mechanisms mediating hNSC homing via the vasculature differ from the mechanisms mediating homing through parenchyma, but also that each step invokes a distinct pathway mediating a specialized function in the hNSC homing cascade. (TNF‐α stimulation also upregulates vascular cell adhesion molecule‐1 expression on the hNSCs themselves and increases NSC‐endothelial interactions.) The selective use of integrin subgroups to mediate homing of cells of neuroectodermal origin may also be used to ensure that cells within the systemic circulation are delivered to the pathological region of a given organ to the exclusion of other, perhaps undesired, organs.

Cell Transplantation and Gene Therapy in Parkinson's Disease

Parkinsons disease is a progressive neurodegenerative disorder affecting, in part, dopaminergic motor neurons of the ventral midbrain and their terminal projections that course to the striatum. Symptomatic strategies focused on dopamine replacement have proven effective at remediating some motor symptoms during the course of disease but ultimately fail to deliver long-term disease modification and lose effectiveness due to the emergence of side effects. Several strategies have been experimentally tested as alternatives for Parkinsons disease, including direct cell replacement and gene transfer through viral vectors. Cellular transplantation of dopamine-secreting cells was hypothesized as a substitute for pharmacotherapy to directly provide dopamine, whereas gene therapy has primarily focused on restoration of dopamine synthesis or neuroprotection and restoration of spared host dopaminergic circuitry through trophic factors as a means to enhance sustained controlled dopamine transmission. This seems now to have been verified in numerous studies in rodents and nonhuman primates, which have shown that grafts of fetal dopamine neurons or gene transfer through viral vector delivery can lead to improvements in biochemical and behavioral indices of dopamine deficiency. However, in clinical studies, the improvements in parkinsonism have been rather modest and variable and have been plagued by graft-induced dyskinesias. New developments in stem-cell transplantation and induced patient-derived cells have opened the doors for the advancement of cell-based therapeutics. In addition, viral-vector-derived therapies have been developed preclinically with excellent safety and efficacy profiles, showing promise in clinical trials thus far. Further progress and optimization of these therapies will be necessary to ensure safety and efficacy before widespread clinical use is deemed appropriate.

Functional Multipotency of Stem Cells: A Conceptual Review of Neurotrophic Factor-Based Evidence and Its Role in Translational Research

We here propose an updated concept of stem cells (SCs), with an emphasis on neural stem cells (NSCs). The conventional view, which has touched principally on the essential property of lineage multipotency (e.g., the ability of NSCs to differentiate into all neural cells), should be broadened to include the emerging recognition of biofunctional multipotency of SCs to mediate systemic homeostasis, evidenced in NSCs in particular by the secretion of neurotrophic factors. Under this new conceptual context and taking the NSC as a leading example, one may begin to appreciate and seek the “logic” behind the wide range of molecular tactics the NSC appears to serve at successive developmental stages as it integrates into and prepares, modifies, and guides the surrounding CNS micro- and macro-environment towards the formation and self-maintenance of a functioning adult nervous system. We suggest that embracing this view of the “multipotency” of the SCs is pivotal for correctly, efficiently, and optimally exploiting stem cell biology for therapeutic applications, including reconstitution of a dysfunctional CNS.

Microarray-based Transcriptional and Epigenetic Profiling of Matrix Metalloproteinases, Collagens, and Related Genes in Cancer

Epigenetic parameters (DNA methylation, histone modifications, and miRNAs) play a significant role in cancer. To identify the common epigenetic signatures of both the individual matrix metalloproteinases (MMPs) and the additional genes, the function of which is also linked to proteolysis, migration, and tumorigenesis, we performed epigenetic profiling of 486 selected genes in unrelated non-migratory MCF-7 breast carcinoma and highly migratory U251 glioma cells. Genome-wide transcriptional profiling, quantitative reverse transcription-PCR, and microRNA analyses were used to support the results of our epigenetic studies. Transcriptional silencing in both glioma and breast carcinoma cells predominantly involved the repressive histone H3 Lys-27 trimethylation (H3K27me3) mark. In turn, epigenetic stimulation was primarily performed through a gain in the histone H3 Lys-4 dimethylation (H3K4me2) and H3 hyperacetylation and by a global reduction of H3K27me3. Inactive pro-invasive genes in MCF-7 cells but not in U251 cells frequently exhibited a stem cell-like bivalent mark (enrichment in both H3K27me3 and H3K4me2), a characteristic of developmental genes. In contrast with other MMPs, MMP-8 was epigenetically silenced in both cell types, thus providing evidence for the strict epigenetic control of this anti-tumorigenic proteinase in cancer. Epigenetic stimulation of multiple collagen genes observed in cultured glioma cells was then directly confirmed using orthotopic xenografts and tumor specimens. We suggest that the epigenetic mechanisms allow gliomas to deposit an invasion-promoting collagen-enriched matrix and then to use this matrix to accomplish their rapid migration through the brain tissue.

Large animal models are critical for rationally advancing regenerative therapies

‘While such ‘small-animal’ systems offer invaluable insights into fundamental biological questions, it is often misleading and perilous to unquestionably equate the higher order motor, sensory and cognitive processes that characterize human disease with that gleaned from a mouse or rat’ Enthusiasm for therapies based on the transplantation of exogenous cells or the transfer of genes by viral vectors has burgeoned over the past 30 years, accompanied by a predictable exhortation to launch clinical trials as soon as possible. Most data regarding safety, efficacy and mechanisms of these therapies have been derived from studies in rodents alone. While such ‘small-animal’ systems offer invaluable insights into fundamental biological questions, it is often misleading and perilous to unquestionably equate the higher order motor, sensory and cognitive processes that characterize human disease with that gleaned from a mouse or rat. Indeed, the literature is littered with clinical trials that failed and, in some cases, led to unforeseen adverse outcomes because the field leap-frogged over the requisite large-animal model. Large animals often provide an essential bridge between insights into fundamental biology and pathophysiology gleaned from simple systems and the realities of treating a human disease. Often, this is especially true for neurological disorders where not only differences in size and scale pertain, but also in neuroanatomical connections and organization, cognitive capacities, signaling pathways, genetic redundancy or the disease etiology. While the gene therapy field has increased their use of nonhuman primates prior to the application of viral vectors in clinical trials, the cellular therapy field – represented most conspicuously of late by the stem cell field – has only recently begun to properly address this requirement. Monkeys and the minipig may prove to be excellent preclinical models owing to their similar comparative anatomy, pharmacokinetics and physiological and metabolic

Human Neural Stem Cells Survive Long Term in the Midbrain of Dopamine-Depleted Monkeys After GDNF Overexpression and Project Neurites Toward an Appropriate Target

Transplanted multipotent human fetal neural stem cells (hfNSCs) significantly improved the function of parkinsonian monkeys in a prior study primarily by neuroprotection, with only 3%–5% of cells expressing a dopamine (DA) phenotype. In this paper, we sought to determine whether further manipulation of the neural microenvironment by overexpression of a developmentally critical molecule, glial cell‐derived neurotrophic factor (GDNF), in the host striatum could enhance DA differentiation of hfNSCs injected into the substantia nigra and elicit growth of their axons to the GDNF‐expressing target. hfNSCs were transplanted into the midbrain of 10 green monkeys exposed to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydro‐pyridine. GDNF was delivered concomitantly to the striatum via an adeno‐associated virus serotype 5 vector, and the fate of grafted cells was assessed after 11 months. Donor cells remained predominantly within the midbrain at the injection site and sprouted numerous neurofilament‐immunoreactive fibers that appeared to course rostrally toward the striatum in parallel with tyrosine hydroxylase‐immunoreactive fibers from the host substantia nigra but did not mature into DA neurons. This work suggests that hfNSCs can generate neurons that project long fibers in the adult primate brain. However, in the absence of region‐specific signals and despite GDNF overexpression, hfNSCs did not differentiate into mature DA neurons in large numbers. It is encouraging, however, that the adult primate brain appeared to retain axonal guidance cues. We believe that transplantation of stem cells, specifically instructed ex vivo to yield DA neurons, could lead to reconstruction of some portion of the nigrostriatal pathway and prove beneficial for the parkinsonian condition.

Neonatal immune-tolerance in mice does not prevent xenograft rejection.

Assessing the efficacy of human stem cell transplantation in rodent models is complicated by the significant immune rejection that occurs. Two recent reports have shown conflicting results using neonatal tolerance to xenografts in rats. Here we extend this approach to mice and assess whether neonatal tolerance can prevent the rapid rejection of xenografts. In three strains of neonatal immune-intact mice, using two different brain transplant regimes and three independent stem cell types, we conclusively show that there is rapid rejection of the implanted cells. We also address specific challenges associated with the generation of humanized mouse models of disease.

Cellular Repair in the Parkinsonian Nonhuman Primate Brain

Parkinson disease (PD) is a neurodegenerative disorder that provides a useful model for testing cell replacement strategies to rejuvenate the affected dopaminergic neural systems, which have been destroyed by aging and the disease. We first showed that grafts of fetal dopaminergic neurons can reverse parkinsonian motor deficits induced by the toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), validating the feasibility of cellular repair in a primate nervous system. Subsequent clinical trials in Parkinson patients showed encouraging results, including long-term improvement of neurological signs and reduction of medications in some patients. However, many experienced little therapeutic benefit, and some recipients experienced dyskinesias, suggesting a lack of regulated control of the grafts. We have since attempted to improve cell replacements by placing grafts in their correct anatomical location in the substantia nigra and using strategies such as co-grafting fetal striatal tissue or growth factors into the physiologic striatal targets. Moreover, the use of fetal cells depends on a variable supply of donor material, making it difficult to standardize cell quality and quantity. Therefore, we have also explored possibilities of using human neural stem cells (hNSCs) to ameliorate parkinsonism in nonhuman primates with encouraging results. hNSCs implanted into the striatum showed a remarkable migratory ability and were found in the substantia nigra, where a small number appeared to differentiate into dopamine neurons. The majority became growth factor-producing glia that could provide beneficial effects on host dopamine neurons. Studies to determine the optimum stage of differentiation from embryonic stem cells and to derive useful cells from somatic cell sources are in progress.