Long-term fate of neural precursor cells following transplantation into developing and adult CNS

Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can serve as an effective and safe replacement therapy for CNS injury and degeneration.     

Survival of neural progenitor cells from the subventricular zone of the adult rat after transplantation into the host spinal cord of the same strain of adult rat

The subventricular zone (SVZ) of the lateral ventricle of the mammalian forebrain is the major site in which neural progenitor cells (NPC) persist in the adult brain. The NPC are located beneath ventricular ependymal cells and have the capacity to self-renew and continuously produce neurons and glial cells. We have shown previously that neurospheres can be obtained from the brain of deceased adult rats and that neurosphere cells survive after transplantation into the spinal cord. In the present study, we investigated whether fresh NPC from living adult rats can survive and be integrated into host tissues after transplantation into the adult rat spinal cord of the same strain. We used rats expressing transgenic green fluorescent protein (GFP) as a donor to identify the transplanted NPCs. The SVZ tissues were obtained from the striatal wall of the lateral ventricle of adult GFP-rats and were grafted into lesions of the spinal cord at the cervical level. Two to 3 weeks after grafting, NPC migrated through the host tissue 0.5-1 mm away from the implantation site, and were integrated into the white matter of the host spinal cord. Surviving NPC exhibited immunohistochemical phenotypes of astrocytes (glial fibrillary acidic protein), but not for neurons (alpha-tubulin III) or oligodendrocytes (Rip; Hybridoma Bank, Iowa City, IA, USA). Thus, NPC from the SVZ of adult rats can survive and differentiate into at least astrocytes, which can then be integrated into host tissue after transplantation into spinal cord lesions in the adult rat.     

Multipotent and restricted precursors in the central nervous system.

Acquisition of cell type-specific properties in the nervous system is likely a process of sequential restriction in developmental potential. At least two classes of pluripotent stem cells, neuroepithelial (NEP) stem cells and EGF-dependent neurosphere stem cells, have been identified in distinct spatial and temporal domains. Pluripotent stem cells likely generate central nervous system (CNS) and peripheral nervous system (PNS) derivatives via the generation of intermediate lineage-restricted precursors that differ from each other and from multipotent stem cells. Neuronal precursors termed neuronal-restricted precursors (NRPs), multiple classes of glial precursors termed glial-restricted precursors (GRPs), oligodendrocyte-type 2 astrocytes (O2As), astrocyte precursor cells (APCs), and PNS precursors termed neural crest stem cells (NCSCs) have been identified. Multipotent stem cells and restricted precursor cells can be isolated from embryonic stem (ES) cell cultures providing a non-fetal source of such cells. Analysis in multiple species illustrates similarities between rat, mouse, and human cell differentiation raising the possibility that similar factors and markers may be used to isolate precursor cells from human tissue or ES cells. Anat Rec (New Anat): 257:137-143, 1999.     

Single neural progenitor cells derived from EGFP expressing mice is useful after spinal cord injury in mice

Neural stem cells (NSCs) were widely used for studying the cell's replacement after transplantation in nervous system because of its specific characteristics. However, Stracing the cells after transplantation was still a problem. In the present study, we isolated and cultured the neural stem cells from the C57BL/6J EGFP transgenic mouse (EGFP mice), and identified the capacity for self-renewal and differentiation into the three CNS lineages (neurons, astrocytes, and oligodendrocytes). Then we transplanted the single neural stem cell into the lesion spinal cord. Expression of GFP and differentiation was evaluated at two weeks post-transplantation. The data showed that these neural stem cells derived from the EGFP mice could maintain transgene expression and could differentiate into the MAP2 positive cells after transplantation into the injured spinal cord. The results suggested that NSC expressing EGFP was a useful marker for tracing the cells after transplantation in vivo and functional in the treatment to spinal cord injury.     

Reestablishment of damaged adult motor pathways by grafted embryonic cortical neurons

Damage to the adult motor cortex leads to severe and frequently irreversible deficits in motor function. Transplantation of embryonic cortical neurons into the damaged adult motor cortex was previously shown to induce partial recovery, but reports on graft efferents have varied from no efferent projections to sparse innervation. Here, we grafted embryonic cortical tissue from transgenic mice overexpressing a green fluorescent protein into the damaged motor cortex of adult mice. Grafted neurons developed efferent projections to appropriate cortical and subcortical host targets, including the thalamus and spinal cord. These projections were not a result of cell fusion between the transplant and the host neurons. Host and transplanted neurons formed synaptic contacts and numerous graft efferents were myelinated. These findings demonstrate that there is substantial anatomical reestablishment of cortical circuitry following embryonic cortex grafting into the adult brain. They suggest that there is an unsuspected potential for neural cell transplantation to promote reconstruction after brain injury.     

Immunohistochemical Study of Fetal Stem/Progenitor Cells from Human Brain Transplanted into Traumatized Spinal Cord of Adult Rats

Neural stem/progenitor cells from human fetal brain were grown in a tissue culture and transplanted into traumatized spinal cord of adult rats. The behavior and differentiation of transplanted cells were studied morphologically by means of histological and immunohistochemical methods and confocal microscopy. Human neural stem/progenitor cells were viable for not less than 3 months. They migrated and differentiated into neurons and glia in the traumatized spinal cord of adult rats.     

Xenotransplantation of stem/progenitor cells from human fetal brain to adult rats with spinal trauma

In vitro grown neural stem cells from human fetal brain were transplanted to adult rats with spinal trauma. The spinal cord was examined morphologically using histological and immunohistochemical methods on days 5, 15, 30, and 110. Human neural stem/progenitor cells were viable, migrated, and differentiated into neurons and glia in the traumatized spinal cord in adult rats.     

Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat

Pluripotent or multipotent stem cells isolated from human embryos or adult central nervous system (CNS) may provide new neurons to ameliorate neural disorders. A major obstacle, however, is that the majority of such cells do not differentiate into neurons when grafted into non-neurogenic areas of the adult CNS. Here we report a new in vitro priming procedure that generates a nearly pure population of neurons from fetal human neural stem cells (hNSCs) transplanted into adult rat CNS. Furthermore, the grafted cells differentiated by acquiring a cholinergic phenotype in a region-specific manner. This technology may advance stem cell-based therapy to replace lost neurons in neural injury or neurodegenerative disorders.     

Focal transplantation-based astrocyte replacement is neuroprotective in a model of motor neuron disease

Cellular abnormalities in amyotrophic lateral sclerosis (ALS) are not limited to motor neurons. Astrocyte dysfunction also occurs in human ALS and transgenic rodents expressing mutant human SOD1 protein (SOD1(G93A)). Here we investigated focal enrichment of normal astrocytes using transplantation of lineage-restricted astrocyte precursors, called glial-restricted precursors (GRPs). We transplanted GRPs around cervical spinal cord respiratory motor neuron pools, the principal cells whose dysfunction precipitates death in ALS. GRPs survived in diseased tissue, differentiated efficiently into astrocytes and reduced microgliosis in the cervical spinal cords of SOD1(G93A) rats. GRPs also extended survival and disease duration, attenuated motor neuron loss and slowed declines in forelimb motor and respiratory physiological functions. Neuroprotection was mediated in part by the primary astrocyte glutamate transporter GLT1. These findings indicate the feasibility and efficacy of transplantation-based astrocyte replacement and show that targeted multisegmental cell delivery to the cervical spinal cord is a promising therapeutic strategy for slowing focal motor neuron loss associated with ALS.     

培养胚胎神经干细胞移植入纹状体后的形态学观察

本研究的目的是 :将体外培养的小鼠胚胎大脑皮层和脊髓的神经干细胞经单细胞悬液微移植后观察其在大鼠纹状体的存活、迁移和分化的状况。实验在无血清条件下将这些细胞扩增、培养再经活细胞荧光染料 Di I标记后 ,采用微移植的方法 ,通过脑立体定位仪上用微玻璃针将干细胞分别植入成年大鼠双侧纹状体的对称部位。大鼠存活 8周后 ,经灌注固定、恒冷箱切片 ,在荧光显微镜下观察标记的移植细胞的迁移状况 ;用星形胶质细胞特异性抗体观察移植区 GFAP的表达 ,以显示移植细胞分化状况。结果表明 :来源于胚胎小鼠大脑皮层和脊髓的体外培养神经干细胞经微移植后 ,均可在成年大鼠脑内纹状体区域存活 ,移植的神经干细胞可向周围的脑实质内迁移 ,迁移细胞沿特定的纹状体结构分布。神经干细胞主要分化成星形胶质细胞。本实验结果提示 ,移植的神经干细胞可在脑实质内存活、迁移和分化。     

Remyelination by transplanted oligodendrocytes of a demyelinated lesion in the spinal cord of the adult shiverer mouse

Fragments of normal newborn mouse central nervous system (CNS) were transplanted into the spinal cord of adult shiverer mice at distance of 1, 2 or 3 intervertebral spaces from a lysolecithin-induced demyelinating lesion. Remyelination by grafted oligodendrocytes was observed by electron microscopy (EM). This result showed the capability of grafted oligodendrocytes or precursor cells to migrate to a demyelinated lesion and to remyelinate naked axons in an adult host, even in presence of host spontaneous remyelination.     

Robust neural integration from retinal transplants in mice deficient in GFAP and vimentin

With recent progress in neuroscience and stem-cell research, neural transplantation has emerged as a promising therapy for treating CNS diseases. The success of transplantation has been limited, however, by the restricted ability of neural implants to survive and establish neuronal connections with the host. Little is known about the mechanisms responsible for this failure. Neural implantation triggers reactive gliosis, a process accompanied by upregulation of intermediate filaments in astrocytes and formation of astroglial scar tissue. Here we show that the retinas of adult mice deficient in glial fibrillary acidic protein and vimentin, and consequently lacking intermediate filaments in reactive astrocytes and Müller cells, provide a permissive environment for grafted neurons to migrate and extend neurites. The transplanted cells integrated robustly into the host retina with distinct neuronal identity and appropriate neuronal projections. Our results indicate an essential role for reactive astroglial cells in preventing neural graft integration after transplantation.     

胚胎脊髓移植在成鼠损伤脊髓神经通路重建中的作用——电镜CB—HRP示踪

目的：探讨胎鼠脊髓在成鼠损伤脊髓神经通路修复中的作用。方法：将Ｅ１４大鼠胚胎脊髓植入成鼠损伤脊髓后３０、５０、７０天时，通过坐骨神经和红核引入ＣＢ－ＨＲＰ，取移植部位脊髓做冰冻切片，经ＴＭＢ组化显色后，再分步制成电镜标本，然后在电镜下观察移植物和宿主脊髓的纤维联系。结果：术后３０天时，来自背根的轴突再生进入移植物，附近运动神经元和中间神经元的突起此时向移植物内发送新支。术后５０天时，来自背根有的轴     

Autologous adult rodent neural progenitor cell transplantation represents a feasible strategy to promote structural repair in the chronically injured spinal cord

Adult neural progenitor cells (NPCs) represent an attractive source for cell-based regenerative strategies in CNS disease. In animal models of spinal cord injury, syngenic adult NPCs, which were isolated from pooled post-mortem CNS tissue and co-transplanted together with fibroblasts, have been shown to promote substantial structural repair. The autologous transplantation of adult NPCs represents a major advantage compared with other sources of neural stem/progenitor cells. However, the feasibility of autologous NPC generation from a single biopsy in a relevant preclinical CNS disease model has yet to be demonstrated. To investigate this matter, adult Wistar rats underwent a cervical spinal cord lesion, which was followed by a minimal subventricular zone aspiration biopsy 2 days later. NPCs were isolated and propagated separately for each animal for the following 8 weeks. Thereafter, they were co-transplanted with simultaneously harvested skin fibroblasts in an autologous fashion into the cervical spinal cord lesion site. A total of 4 weeks later, graft survival, tissue replacement and axonal regeneration were assessed histologically. Animals receiving either allogenic NPCs combined with fibroblasts or autologous pure fibroblast grafts served as control groups. Within 8 weeks after the biopsy more than 3 million NPCs could be generated from a single aspiration biopsy, which displayed a differentiation pattern indistinguishable from syngenic NPC grafts. NPCs within autologous co-grafts readily survived, replaced cystic lesion defects completely and differentiated exclusively into glial phenotypes, thus paralleling previous findings with syngenic NPCs. The delayed transplantation 8 weeks after the spinal cord lesion elicited substantial axonal regeneration. These findings demonstrate that the therapeutic strategy to induce structural repair by transplanting adult autologous NPCs, after the successful propagation from a small brain biopsy into an acute CNS disease model, such as spinal cord injury, is feasible at the preclinical level.     

Mesencephalic human neural progenitor cells transplanted into the adult hemiparkinsonian rat striatum lack dopaminergic differentiation but improve motor behavior

The clinical outcome of cell replacement therapies depends upon the successful survival and differentiation of transplanted cells. Here, we transplanted human neural progenitor cells derived from the ventral mesencephalon of an 8-week-old embryo into the ipsilateral (right) striatum of unilateral 6-hydroxydopamine-lesioned adult rats. To assess the therapeutic potency of grafted cells, 2 independent behavioral tests were conducted 12 weeks after transplantation: in the rotation test, a mild behavioral improvement was detected, and in the cylinder test, transplanted animals overcame the lesion-induced right forepaw preference. To address this behavioral improvement to a dopaminergic differentiation capacity of transplanted cells in vivo, immunohistochemistry for tyrosine hydroxylase was performed, showing a total lack of immunoreactivity. However, we found a considerable number of transplanted human nuclei-positive cells preferentially differentiated into neurons. In addition, glial fibrillary acidic protein-expressing cells were also detected. Our results show that behavioral improvement does not necessarily correlate with a differentiation of transplanted precursors into dopaminergic neurons, indicating other factors to be involved in a partial functional recovery. Nevertheless, for the development of a clinically useful cell therapy, it is important to overcome obstacles, namely the poor dopaminergic differentiation of human neural progenitor cells after grafting.     

Fictive motor activities in adult chronic spinal rats transplanted with embryonic brainstem neurons

The present study was designed to examine the effects of an intraspinal transplantation of embryonic brainstem neurons on fictive motor patterns which can develop in hindlimb nerves of adult chronic spinal rats. Seventeen adult rats were spinalized at T8-9 level and, 8 days later, a suspension of embryonic cells obtained either from the raphe region (RR, n=8) or from the locus coeruleus (LC, n=9) was injected caudally (T12–13) to the cord transection. Eight control animals (control rats) were spinalized and injected with vehicle under the same conditions. One to three months later, the animals were decorticated and fictive motor patterns were recorded in representative hindlimb nerves. The data revealed that both control and grafted spinal rats could exhibit two distinctly different fictive motor patterns, one which could be associated with stepping and the other with hindlimb paw shaking. They further showed that following transplantation of embryonic RR or LC neurons the excitability of the spinal stepping generator was increased, whereas that of the spinal neural circuits which generate hindlimb paw shaking was not significantly affected. A histological analysis performed on the spinal cord segments below the transection revealed complete absence of serotonin and noradrenaline immunoreactivity in control spinal animals and, in both types of grafted rats, an extensive monoaminergic reinnervation with synaptic contacts between monoaminergic transplanted neurons and host interneurons and/or motoneurons. The possible mechanisms by which grafted monoaminergic neurons can influence the spinal motor networks are discussed.     

Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats

Human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for transplantation. In Parkinson's disease, hESC-derived dopaminergic (DA) neurons may replace the degenerated neurons in the brain. Here, we generated highly enriched cultures of neural progenitors from hESCs and grafted the progenitors into the striatum of Parkinsonian rats. The grafts survived for at least 12 weeks, the transplanted cells stopped proliferating, and teratomas were not observed. The grafted cells differentiated in vivo into DA neurons, though at a low prevalence similar to that observed following spontaneous differentiation in vitro. Transplanted rats exhibited a significant partial correction of D-amphetamine and apomorphine-induced rotational behavior, along with a significant improvement in stepping and placing non-pharmacological behavioral tests. While transplantation of uncommitted hESC-derived neural progenitors induced partial behavioral recovery, our data indicate that the host-lesioned striatum could not direct the transplanted neural progenitors to acquire a dopaminergic fate. Hence, induction of their differentiation toward a midbrain fate prior to transplantation is probably required for complete correction of behavioral deficit. Our observations encourage further developments for the potential use of hESCs in the treatment of Parkinson's disease.     

Long-term Fate of Allogeneic Neural Stem Cells Following Transplantation into Injured Spinal Cord

To characterize the fate of allogeneic neural stem cells (NSCs) following transplantation into injured spinal cord, green fluorescent protein (GFP)-NSCs isolated from GFP transgenic Sprague–Dawley rat embryos were transplanted into contused spinal cords of Wistar rats. The GFP-NSCs survived for at least 6 months in injured spinal cord; most of them differentiated rapidly into astrocytes, and a few were able to undergo proliferation. After transplantation, the GFP-NSCs remained in the transplantation site at the early stage, and then migrated along white-matter, and gathered around the injured cavity. At 6 months post-transplantation, CD8 T-lymphocytes infiltrated the spinal cord, and mixed lymphocyte culture from host and donor showed that lymphocytes from the host spleen were primed by allogeneic GFP-NSCs. At 12 months post-transplantation, most GFP cells in the spinal cord lost their morphology and disintegrated. The Basso, Beattie and Bresnahan score and footprint analysis indicated that the improvement of locomotor function in transplanted rats appeared only at the early stage, and was not seen even at 6 months after transplantation All these results suggest that the allogeneic NSCs, after transplantation into injured spinal cord, activate the host immune system. Therefore, if immunosuppressive agents are not used, the grafted allogeneic NSCs, although they can survive for a long time, are subjected to host immune rejection, and the effect of NSCs on functional recovery is limited.     

神经干细胞移植对小鼠脊髓损伤的影响

目的观察神经干细胞(NSCs)移植对脊髓损伤(SCI)小鼠功能恢复的影响。方法分离、培养、增殖和纯化E14-17d小鼠的NSCs,并通过免疫荧光法对其进行鉴定。将绿色荧光蛋白转基因小鼠的NSCs移植到小鼠脊髓损伤模型体内,术后进行行为学和病理学检测,观察小鼠功能恢复情况。运用改良Allen's法制备小鼠T10-T11脊髓损伤动物模型,动物分为假手术组(12只)、模型组(12只),治疗组(12只)和对照组6(12只)。治疗组每只小鼠自眶静脉注射NSCs悬液200μl(2×10个细胞),对照组只注射DMEM/F12培养基200μl。术后1d、3d、7d、14d、21d、28d和56d,进行BBB运动功能评分和病理学检测,观察植入区细胞生长变化情况。结果 BBB评分显示:治疗组明显高于对照组(<0.01),治疗组与假手术组相比没有明显差异(>0.05),说明NSCs移植后小鼠的行为学得到了明显改善,功能有所恢复。病理学检测发现,移植后NSCs不仅迁移到脊髓损伤区,而且与宿主细胞较好地整合。结论移植的E14-17d胚胎小鼠NSCs不仅可在脊髓损伤部位存活并和宿主细胞整合,而且可促进小鼠后肢运动功能恢复。