Candidates For Cell Replacement Therapy Research Articles

Comparison of the effects of EGF, FGF-b, and NGF on the proliferation, migration, and reprogramming of primary rat Müller cells.

During the healing process of full-thickness macular holes (FTMHs), the closure and recovery of the hole depend on the migration, proliferation, and activation of Müller cells to promote the closure of holes and restoration of the photosensitive layer. In this study, we investigated the ability of the epidermal growth factor (EGF), fibroblast growth factor-basic (FGF-b), and nerve growth factor (NGF) to influence this process by regulating proliferation, migration, and reprogramming of primary rat Müller cells. Cell proliferation was measured using CCK8 [2- (2-Methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-tetrazolium Sodium Salt] colorimetric assays and EdU [5-Ethynyl-2'-deoxyuridine] assays over 48 h. Cell migration was measured using scratch-wound assays and transwell migration assays over 48 h. In addition, we conducted Western blot assays and immunofluorescence assays on cells that were specially treated for 1, 3, and 5 days for cell reprogramming. The percentage of EdU-positive cells in Nestin-positive have also been tested by co-immunofluorescence (Co-IF) staining. EGF and FGF-b significantly promoted the proliferation of Müller cells (p < 0.05) at a concentration of 0-50 ng/mL, but NGF did not (p > 0.05), compared to untreated controls. Exogenous FGF-b and EGF promote the reprogramming of primary rat Müller cells, significantly enhancing the neural stem cell marker Nestin after stimulation on the 1st, 3rd, and 5th days, respectively. The expression of Müller cell marker Vimentin was significantly (p < 0.05) reduced during this period compared to the control group. However, there was no significant difference between the NGF and control groups. Furthermore, the EGF group expressed stronger Nestin expression than the SCM group. The Co-IF staining showed that early 50% of activated cells came from newly proliferating cells on the 5th day. These observations suggest that FGF-b can promote the activation of Müller cells in a short time and enhance the possessive features of neural stem cells, while EGF may act for a longer period of time. This may further the understanding of growth factor therapy in treating FTMHs, and Müller glia may be promising candidates for cell replacement therapy.

Combining physical & cognitive training with iPSC-derived dopaminergic neuron transplantation promotes graft integration & better functional outcome in parkinsonian marmosets

Parkinson's disease (PD) is a relentlessly progressive and currently incurable neurodegenerative disease with significant unmet medical needs. Since PD stems from the degeneration of midbrain dopaminergic (DA) neurons in a defined brain location, PD patients are considered optimal candidates for cell replacement therapy. Clinical trials for cell transplantation in PD are beginning to re-emerge worldwide with a new focus on induced pluripotent stem cells (iPSCs) as a source of DA neurons since they can be derived from adult somatic cells and produced in large quantities under current good manufacturing practices. However, for this therapeutic strategy to be realized as a viable clinical option, fundamental translational challenges need to be addressed including the manufacturing process, purity and efficacy of the cells, the method of delivery, the extent of host reinnervation and the impact of patient-centered adjunctive interventions. In this study we report on the impact of physical and cognitive training (PCT) on functional recovery in the nonhuman primate (NHP) model of PD after cell transplantation. We observed that at 6 months post-transplant, the PCT group returned to normal baseline in their daily activity measured by actigraphy, significantly improved in their sensorimotor and cognitive tasks, and showed enhanced synapse formation between grafted cells and host cells. We also describe a robust, simple, efficient, scalable, and cost-effective manufacturing process of engraftable DA neurons derived from iPSCs. This study suggests that integrating PCT with cell transplantation therapy could promote optimal graft functional integration and better outcome for patients with PD.

The functional maturity of grafted human pluripotent stem cell derived-islets (hSC-Islets) evaluated by the glycemic set point during blood glucose normalizing process in diabetic mice

Human pluripotent stem cell (hPSCs) derived-pancreatic islets (hSC-islets) are good candidates for cell replacement therapy for patients with diabetes as substitutes for deceased donor-derived islets, because they are pluripotent and have infinite proliferation potential. Grafted hSC-islets ameliorate hyperglycemia in diabetic mice; however, several weeks are needed to normalize the hyperglycemia. These data suggest hSC-islets require maturation, but their maturation process in vivo is not yet fully understood. In this study, we utilized two kinds of streptozotocin (STZ)-induced diabetes model mice by changing the administration timing in order to examine the time course of maturation of hSC-islets and the effects of hyperglycemia on their maturation. We found no hyperglycemia in immune-compromised mice when hSC-islets had been transplanted under their kidney capsules in advance, and STZ was administered 4 weeks after transplantation. Of note, the blood glucose levels of those mice were stably maintained under 100 mg/dl 10 weeks after transplantation; this is lower than the mouse glycemic set point (120–150 mg/dl), suggesting that hSC-islets control blood glucose levels to the human glycemic set point. We confirmed that gene expression of maturation markers of pancreatic beta cells tended to upregulate during 4 weeks after transplantation. Periodical histological analysis revealed that revascularization was observed as early as 1 week after transplantation, but reinnervation in the grafted hSC-islets was not detected at all, even 15 weeks after transplantation. In conclusion, our hSC-islets need at least 4 weeks to mature, and the human glycemic set point is a good index for evaluating ultimate maturity for hSC-islets in vivo.

Electric field stimulation boosts neuronal differentiation of neural stem cells for spinal cord injury treatment via PI3K/Akt/GSK-3β/β-catenin activation

BackgroundNeural stem cells (NSCs) are considered as candidates for cell replacement therapy in many neurological disorders. However, the propensity for their differentiation to proceed more glial rather than neuronal phenotypes in pathological conditions limits positive outcomes of reparative transplantation. Exogenous physical stimulation to favor the neuronal differentiation of NSCs without extra chemical side effect could alleviate the problem, providing a safe and highly efficient cell therapy to accelerate neurological recovery following neuronal injuries.ResultsWith 7-day physiological electric field (EF) stimulation at 100 mV/mm, we recorded the boosted neuronal differentiation of NSCs, comparing to the non-EF treated cells with 2.3-fold higher MAP2 positive cell ratio, 1.6-fold longer neuronal process and 2.4-fold higher cells ratio with neuronal spontaneous action potential. While with the classical medium induction, the neuronal spontaneous potential may only achieve after 21-day induction. Deficiency of either PI3Kγ or β-catenin abolished the above improvement, demonstrating the requirement of the PI3K/Akt/GSK-3β/β-catenin cascade activation in the physiological EF stimulation boosted neuronal differentiation of NSCs. When transplanted into the spinal cord injury (SCI) modelled mice, these EF pre-stimulated NSCs were recorded to develop twofold higher proportion of neurons, comparing to the non-EF treated NSCs. Along with the boosted neuronal differentiation following transplantation, we also recorded the improved neurogenesis in the impacted spinal cord and the significantly benefitted hind limp motor function repair of the SCI mice.ConclusionsIn conclusion, we demonstrated physiological EF stimulation as an efficient method to boost the neuronal differentiation of NSCs via the PI3K/Akt/GSK-3β/β-catenin activation. Pre-treatment with the EF stimulation induction before NSCs transplantation would notably improve the therapeutic outcome for neurogenesis and neurofunction recovery of SCI.

Human olfactory epithelium-derived stem cells ameliorate histopathological deficits and improve behavioral functions in a rat model of cerebellar ataxia

Cell replacement therapy (CRT) is one of the most effective approaches used to alleviate symptoms of neurodegenerative syndromes such as cerebellar ataxia (CA). Human olfactory epithelium mesenchymal stem cells (OE-MSCs) have been recognized as a promising candidate for CRT, due to their distinctive features including immunomodulatory properties and ease of accessible compared to other types of MSCs. Hence, the main goal of our study was to explore the impacts of OE-MSCs transplantation on behavioral, structural, and histological deficiencies in a rat model of CA. After obtained an informed consent from volunteers, OE-MSCs were obtained from their nasal cavity. Then, OE-MSCs were characterized by the positive expression of CD73, CD90, and CD105 as MSCs as well as nestin and vimentin as primitive neuroectodermal stem cells markers. Then, the animals were randomized into three control, 3-acetylpyridine (3-AP) treated, and 3-AP + cell groups. In both experimental groups, the rats received intraperitoneal injection of 3-AP (75 mg/kg), followed by the implantation of OE-MSCs into the cerebellum of 3-AP + cell group. The impact of engrafted OE-MSCs on motor coordination and performance along with biochemical, immunohistochemical, and stereological changes in the cerebellum of the rat models of CA were investigated. According to our findings, the administration of 3-AP decreased the cerebellar GSH concentration. The injection of 3-AP also altered the morphological characteristics of the cerebellar Golgi cells. On the other hand, OE‐MSCs transplantation improved motor coordination in CA. Besides, the implantation of OE‐MSCs reduced caspase-3 expression and microglia proliferation in the cerebellum upon 3-AP administration. Finally, the transplant of OE‐MSCs protected Purkinje cells against 3-AP toxicity. In sum, the present study revealed considerable advantages of OE-MSCs in managing CA animal model.

Neonatal Transplant in Hypoxic Injury.

Even with therapeutic hypothermia as standard of care, hypoxic-ischemic encephalopathy in neonates often causes long-term disabilities. Stem cell therapy may be a successful treatment for HIE both in the acute and chronic time periods post-injury. Neurogenic astrocytes with characteristics of neural stem cells can be cultured as adherent monolayers and be induced to generate neuronal progeny in vitro and in vivo following reintroduction into the neural stem cell niche of both neonatal and adult hosts. Thus, these neurogenic astrocytes represent promising candidates for cell replacement therapy in HIE. Such an approach requires optimized cell cultivation protocols as well as extensive testing of donor cells to assess their capacity for engraftment, survival, and integration in the HIE animal models. In this chapter, we will describe methods of generating the HIE model, generating and culturing monolayer neurogenic astrocytes, and transplanting these cells into HIE animal models.

Generation of a heterozygous GAPDH-Luciferase human ESC line (HVRDe008-A-1) for in vivo monitoring of stem cells and their differentiated progeny

Human stem cell-derived beta (SC-β) cells are a candidate for cell replacement therapy for type 1 diabetes. Whilst refinements to the differentiation protocol have resulted in the production of SC-β cells that resemble adult beta cells, the unsolved challenge to protect transplanted SC-β cells from the host immune system remains. To monitor the survival of SC-β cells in vivo, we knocked-in the Firefly luciferase gene into the GAPDH locus of the HUES8 human embryonic stem cell (hESC) line, such that differentiated islet cells constitutively express luciferase.

Fingolimod increases oligodendrocytes markers expression in epidermal neural crest stem cells

Epidermal neural crest stem cells (EPI-NCSCs) are propitious candidates for cell replacement therapy and supplying neurotrophic factors in the neurological disorders. Considering the potential remyelinating and regenerative effects of fingolimod, in this study, we evaluated its effects on EPI-NCSCs viability and the expression of neurotrophic and oligodendrocyte differentiation factors. EPI-NCSCs, extracted from the bulge of rat hair follicles, were characterized and treated with fingolimod (0, 50, 100, 200, 400, 600, 1000, and 5000 nM). The cell viability was evaluated by MTT assay at 6, 24 and 72 h. The expression of neurotrophic and differentiation factors in the cells treated with 100 and 400 nM fingolimod were measured at 24 and 120 h. Fingolimod at 50–600 nM increased the cells viability after 6 h, with no change at the higher concentrations. The highest concentration (5000nM) induced toxicity at 24 and 72 h. NGF and GDNF genes expression were decreased at 120 h, but on the contrary, brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) were increased by both concentrations at both time points. Oligodendrocyte markers including platelet-derived growth factor receptor A (PDGFRα), neuron-glial antigen 2 (NG2) and growth associated protein 43 (GAP43) were elevated at 120 h, which was accompanied with reduce in stemness markers (Nestin and early growth response 1 (EGR1)). Fingolimod increased the expression of neurotrophic factors in EPI-NCSCs, and guided them to oligodendrocyte fate. Therefore, fingolimod in combination with EPI-NCSCs, can be considered as a promising approach for demyelinating neurological disorders.

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies.

Parkinson's disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

SummaryDefects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.

Comparison of Three Types of Mesenchymal Stem Cells (Bone Marrow, Adipose Tissue, and Umbilical Cord-Derived) as Potential Sources for Inner Ear Regeneration.

In this review, we compared the potential of mesenchymal stem cells derived from bone marrow, adipose tissue and umbilical cord as suitable sources for regeneration of inner ear hair cells and auditory neurons. Our intensive literature search indicates that stem cells in some of adult mammalian tissues, such as bone marrow, can generate new cells under physiological and pathological conditions. Among various types of stem cells, bone marrow-derived mesenchymal stem cells are one of the most promising candidates for cell replacement therapy. Mesenchymal stem cells have been reported to invade the damaged area, contribute to the structural reorganization of the damaged cochlea and improve incomplete hearing recovery. We suggest that bone marrow-derived mesenchymal stem cells would be more beneficial than other mesenchymal stem cells.

Adult Human Peripheral Blood Mononuclear Cells Are Capable of Producing Neurocyte or Photoreceptor-Like Cells That Survive in Mouse Eyes After Preinduction With Neonatal Retina.

This study provides support for the use of peripheral blood mononuclear cells (PBMCs) as a potential source of pluripotent stem cells for treating retinal degeneration. First, this study demonstrated that PBMCs can differentiate into retinal neuron-like cells in vitro and in vivo. Second, some transplanted cells expressed markers for neural progenitors, mature neurons, or photoreceptors at 1, 3, and 6 months after subretinal injection. Finally, this study showed that PBMC transplantation can improve the function of a degenerated retina.

Brief review: cell replacement therapies to treat type 1 diabetes mellitus.

Human embryonic stem cells (hESCs) and induced pluripotent cells (iPSCs) have the potential to differentiate into any somatic cell, making them ideal candidates for cell replacement therapies to treat a number of human diseases and regenerate damaged or non-functional tissues and organs. Key to the promise of regenerative medicine is developing standardized protocols that can safely be applied in patients. Progress towards this goal has occurred in a number of fields, including type 1 diabetes mellitus (T1D). During the past 10 years, significant technological advances in hESC/iPSC biochemistry have provided a roadmap to generate sufficient quantities of glucose-responsive, insulin-producing cells capable of eliminating diabetes in rodents. Although many of the molecular mechanisms underlying the genesis of these cells remain to be elucidated, the field of cell-based therapeutics to treat T1D has advanced to the point where the first Phase I/II trials in humans have begun. Here, we provide a concise review of the history of cell replacement therapies to treat T1D from islet transplantations and xenotranplantation, to current work in hESC/iPSC. We also highlight the latest advances in efforts to employ insulin-producing, glucose-responsive β-like cells derived from hESC as therapeutics.

Heat shock protein 60 affects behavioral improvement in a rat model of Parkinson's disease grafted with human umbilical cord mesenchymal stem cell-derived dopaminergic-like neurons.

Parkinson's disease (PD) is a neurodegenerative disorder that is caused by a loss of dopaminergic (DAergic) neurons in mesencephalic substantia nigra (SN). Human umbilical cord mesenchymal stem cells (hUC-MSCs) are capable of self-renewal and differentiation into multiple cell lineages, including DAergic neurons. Thus, hUC-MSCs could be a promising alternative to compensate for the loss of DAergic neurons in PD. In the current study, hUC-MSCs and hUC-MSCs-derived DAergic-like neurons were transplanted into the striatum and SN of a rat model of PD that is induced by 6-hydroxydopamine (6-OHDA). We evaluated their therapeutic effects on improving rotation behavior in the rat and on modulating the level of heat shock protein 60 (Hsp60) expression in the brain. After transplantation, an amelioration of rotation behavior was observed in rats that underwent cell grafting, and hUC-MSCs-derived DAergic-like neurons were superior to hUC-MSCs at inducing behavioral improvement. Western blot and immunohistochemistry analysis indicated significantly elevated levels of Hsp60 in cell-grafted rats compared to 6-OHDA-lesioned (PD) rats. These results demonstrate that hUC-MSCs-based cell transplantation is potential therapeutic treatment for PD, and hUC-MSCs-derived DAergic-like neurons appear to be favorable candidates for cell replacement therapy in PD. Finally, Hsp60 could be involved in a mechanism of behavioral recovery.

661. Assay Development and Qualification for Human Embryonic Stem Cell (hESC)-Derived Cardiomyocytes Intended for Clinical Studies

Human embryonic stem cell (hESC)-derived cellular products have been considered as promising candidates for cell replacement therapy for multiple degenerative diseases. At the Center of Biomedicine and Genetics (CBG), we have manufactured under cGMP numerous hESC-differentiated products including neuron stem cells (NSC), neuronal progenitor cells (NPC), retinal pigment epithelium (RPE) cells, dopaminergic neurons and cardiomyocytes intended for pre-clinical and clinical studies. Specifically, cardiomyocytes are produced through hESC differentiation procedure utilizing adherent and notably suspension culture systems. Prior to their release for clinical application, cardiomyocytes must be thoroughly tested for their identity and purity. We have developed a systematic approach to characterize hESC-derived cardiomyocytes using assays such as real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. The development and qualification of each assay involve the selection and banking of positive and negative controls, selection and banking of critical reagents, determination of assay conditions, qualification of assay, and generation of assay qualification report and standard operating procedure (SOP). We will also examine a number of critical factors including assay specificity, sensitivity, assay controls, and establishment of assay specifications.

Optimizing Differentiation Protocols for Producing Dopaminergic Neurons from Human Induced Pluripotent Stem Cells for Tissue Engineering Applications: Supplementary Issue: Stem Cell Biology.

Parkinson's disease (PD) is a neurodegenerative disorder that results when the dopaminergic neurons (DNs) present in the substantia nigra necessary for voluntary motor control are depleted, making patients with this disorder ideal candidates for cell replacement therapy. Human induced pluripotent stem cells (hiPSCs), obtained by reprogramming adult cells, possess the properties of pluripotency and immortality while enabling the possibility of patient-specific therapies. An effective cell therapy for PD requires an efficient, defined method of DN generation, as well as protection from the neuroinflammatory environment upon engraftment. Although similar in pluripotency to human embryonic stem cells (hESCs), hiPSCs differentiate less efficiently into neuronal subtypes. Previous work has shown that treatment with guggulsterone can efficiently differentiate hESCs into DNs. Our work shows that guggulsterone is able to derive DNs from hiPSCs with comparable efficiency, and furthermore, this differentiation can be achieved inside three-dimensional fibrin scaffolds that could enhance cell survival upon engraftment.

Establishment of a xeno-free culture system that preserves the characteristics of placenta mesenchymal stem cells.

Although stem cells are promising candidates for cell replacement therapies, the vast majority are derived using animal sera, which has risk of being contaminated by animal viruses or toxins. To overcome these potential problems, we initially established multiple lines of stem cells from first-trimester human placenta (fPMSC), which were cultivated using human follicular fluid (hFF) instead of fetal bovine serum (FBS). FF provides a very important microenvironment for the development of oocytes. No differences were found in the general morphology, growth rate, karyotype, gene and surface expressions between placental MSCs cultured in 5% hFF-supplemented medium (fPMSC-X) or 10% FBS-supplemented medium (fPMSC). Differentiation experiments confirmed similar levels of potency in cells grown in either condition. Since hFF preserved the unique features of the stem cells and is free from potential pathogens, it should be considered as the main culture medium supplement for the propagation of human stem cells for clinical applications.

Generating Cartilage Repair from Pluripotent Stem Cells

The treatment of degeneration and injury of articular cartilage has been very challenging for scientists and surgeons. As an avascular and hypocellular tissue, cartilage has a very limited capacity for self-repair. Chondrocytes are the only cell type in cartilage, in which they are surrounded by the extracellular matrix that they secrete and assemble. Autologous chondrocyte implantation for cartilage defects has achieved good results, but the limited resources and complexity of the procedure have hindered wider application. Stem cells form an alternative to chondrocytes as a source of chondrogenic cells due to their ability to proliferate extensively while retaining the potential for differentiation. Adult stem cells such as mesenchymal stem cells have been differentiated into chondrocytes, but the limitations in their proliferative ability and the heterogeneous cell population hinder their adoption as a prime alternative source for generating chondrocytes. Human embryonic stem cells (hESCs) are attractive as candidates for cell replacement therapy because of their unlimited self-renewal and ability for differentiation into mesodermal derivatives as well as other lineages. In this review, we focus on current protocols for chondrogenic differentiation of ESCs, in particular the chemically defined culture system developed in our lab that could potentially be adapted for clinical application.

CD70–CD27 ligation between neural stem cells and CD4+ T cells induces Fas–FasL-mediated T-cell death

IntroductionNeural stem cells (NSCs) are among the most promising candidates for cell replacement therapy in neuronal injury and neurodegenerative diseases. One of the remaining obstacles for NSC therapy is to overcome the alloimmune response on NSCs by the host.MethodsTo investigate the mechanisms of immune modulatory function derived from the interaction of human NSCs with allogeneic T cells, we examined the immune regulatory effects of human NSCs on allogeneic T cells in vitro.ResultsSignificantly, NSCs induced apoptosis of allogeneic T cells, in particular CD4+ T cells. Interaction of CD70 on NSCs and CD27 on CD4+ T cells mediated apoptosis of T cells. Thus, blocking CD70–CD27 interaction prevented NSC-mediated death of CD4+ T cells.ConclusionsWe present a rational explanation of NSC-induced immune escape in two consecutive stages. First, CD70 constitutively expressed on NSCs engaged CD27 on CD4+ T cells, which induced Fas ligand expression on CD4+ T cells. Second, CD4+ T-cell apoptosis was followed by Fas–Fas ligand interaction in the CD4+ T cells.

The potential for stem cell therapies to have an impact on cerebral palsy: opportunities and limitations

Cerebral palsy (CP) is a chronic childhood disorder described by a group of motor and cognitive impairments and results in a substantial socio-economic burden to the individual, family, and healthcare system. With no effective biological interventions, therapies for CP are currently restricted to supportive and management strategies. Cellular transplantation has been suggested as a putative intervention for neural pathology, as mesenchymal and neural stem cells, as well as olfactory ensheathing glia and Schwann cells, have shown some regenerative and functional efficacy in experimental central nervous system disorders. This review describes the most common cell types investigated and delineates their purported mechanisms in vivo. Furthermore, it provides a cogent summary of both current early-phase clinical trials using neural precursor cells (NPCs) and the state of stem cell therapies for neurodegenerative conditions. Although NPCs are perhaps the most promising candidates for cell replacement therapy in the context of CP, much still remains to be understood regarding safety, efficacy, timing, dose, and route of transplantation, as well as the capacity for combinatorial strategies.