Fetal Central Nervous System Tissue Research Articles

Motor neuron replacement therapy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a motor neuron degenerative disease that is also known as Lou Gehrig’s disease in the United States, Charcot’s disease in France, and motor neuron disease in the UK. The loss of motor neurons causes muscle wasting, paralysis, and eventually death, which is commonly related to respiratory failure, within 3–5 years after onset of the disease. Although there are a limited number of drugs approved for amyotrophic lateral sclerosis, they have had little success at treating the associated symptoms, and they cannot reverse the course of motor neuron degeneration. Thus, there is still a lack of effective treatment for this debilitating neurodegenerative disorder. Stem cell therapy for amyotrophic lateral sclerosis is a very attractive strategy for both basic and clinical researchers, particularly as transplanted stem cells and stem cell-derived neural progenitor/precursor cells can protect endogenous motor neurons and directly replace the lost or dying motor neurons. Stem cell therapies may also be able to re-establish the motor control of voluntary muscles. Here, we review the recent progress in the use of neural stem cells and neural progenitor cells for the treatment of amyotrophic lateral sclerosis. We focus on MN progenitor cells derived from fetal central nervous system tissue, embryonic stem cells, and induced pluripotent stem cells. In our recent studies, we found that transplanted human induced pluripotent stem cell-derived motor neuron progenitors survive well, differentiate into motor neurons, and extend axons into the host white matter, not only in the rostrocaudal direction, but also along motor axon tracts towards the ventral roots in the immunodeficient rat spinal cord. Furthermore, the significant motor axonal extension after neural progenitor cell transplantation in amyotrophic lateral sclerosis models demonstrates that motor neuron replacement therapy could be a promising therapeutic strategy for amyotrophic lateral sclerosis, particularly as a variety of stem cell derivatives, including induced pluripotent stem cells, are being considered for clinical trials for various diseases.

Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury.

The greatest challenge to successful treatment of spinal cord injury is the limited regenerative capacity of the central nervous system and its inability to replace lost neurons and severed axons following injury. Neural stem cell grafts derived from fetal central nervous system tissue or embryonic stem cells have shown therapeutic promise by differentiation into neurons and glia that have the potential to form functional neuronal relays across injured spinal cord segments. However, implementation of fetal-derived or embryonic stem cell-derived neural stem cell therapies for patients with spinal cord injury raises ethical concerns. Induced pluripotent stem cells can be generated from adult somatic cells and differentiated into neural stem cells suitable for therapeutic use, thereby providing an ethical source of implantable cells that can be made in an autologous fashion to avoid problems of immune rejection. This review discusses the therapeutic potential of human induced pluripotent stem cell-derived neural stem cell transplantation for treatment of spinal cord injury, as well as addressing potential mechanisms, future perspectives and challenges.

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation. Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside(+) (GalC) and O4(+) oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation. Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Markers of Pluripotency and Differentiation in Human Neural Precursor Cells Derived from Embryonic Stem Cells and CNS Tissue

Cell transplantation therapies for central nervous system (CNS) deficits such as spinal cord injury (SCI) have been shown to be effective in several animal models. One cell type that has been transplanted is neural precursor cells (NPCs), for which there are several possible sources. We have studied NPCs derived from human embryonic stem cells (hESCs) and human fetal CNS tissue (hfNPCs), cultured as neurospheres, and the expression of pluripotency and neural genes during neural induction and in vitro differentiation. mRNA for the pluripotency markers Nanog, Oct-4, Gdf3, and DNMT3b were downregulated during neural differentiation of hESCs. mRNA for these markers was found in nonpluripotent hfNPC at higher levels compared to hESC-NPCs. However, Oct-4 protein was found in hESC-NPCs after 8 weeks of culture, but not in hfNPCs. Similarly, SSEA-4 and CD326 were only found in hESC-NPCs. NPCs from both sources differentiated as expected to cells with typical features of neurons and astrocytes. The expressions of neuronal markers in hESC-NPCs were affected by the composition of cell culture medium, while this did not affect hfNPCs. Transplantation of hESC-NPC or hfNPC neurospheres into immunodeficient mouse testis or subcutaneous tissue did not result in tumor formation. In contrast, typical teratomas appeared in all animals after transplantation of hESC-NPCs to injured or noninjured spinal cords of immunodeficient rats. Our data show that transplantation to the subcutaneous tissue or the testes of immunodeficient mice is not a reliable method for evaluation of the tumor risk of remaining pluripotent cells in grafts.

Staging and preparation of human fetal striatal tissue for neural transplantation in Huntington's disease.

Transplantation of human fetal central nervous system tissue has been shown to be of benefit in Parkinson's disease, and is currently being explored as a therapeutic option in Huntington's disease. The success of a neural transplant is dependent on a number of factors, including the requirement that donor cells are harvested within a given developmental window and that the cell preparation protocols take account of the biological parameters identified in animal models. Although many of the criteria necessary for a successful neural transplant have been defined in animal models, ultimately they must be validated in human studies, and some issues can only ever be addressed in human studies. Furthermore, because neural transplantation of human fetal tissue is limited to small numbers of patients in any one surgical center, largely due to practical constraints, it is crucial that tissue preparation protocols are clearly defined and reproducible, so that (i) multicenter trials are possible and are based on consistent tissue preparation parameters, and (ii) results between centers can be meaningfully analyzed. Here we describe the preparation of human fetal striatum for neural transplantation in Huntington's disease, and report on the validation of a method for estimating the developmental stage of the fetus based on direct morphometric measurements of the embryonic tissue.

An efficient method for the culturing and generation of neurons and astrocytes from second trimester human central nervous system tissue

The isolation culturing and expansion of human neural progenitors cells has important potential clinical applications in cellular transplantation strategies as well as in developmental studies involving the central nervous system (CNS). This study describes an efficient method to culture neurons and astrocytes as primary cultures, as well as from proliferative progenitor cells derived from second trimester fetal CNS tissue. Second trimester fetal human tissue was mechanically dissociated and subjected to trypsindissociation and trituration. The resulting suspension was passed over a Percoll density gradient. The middle (second) fraction of cells was centrifuged to yield a homogenous population of cells with 80%-90% viability. These cells were either cultured directly on laminin coated dishes with defined medium supplemented with fetal bovine serum or in defined medium supplemented with growth factors including epidermal growth factor, basic fibroblast growth factor and leukemia inhibitory factor. The primary cell cultures yielded neurons and astrocytes after 3-5 days in vitro verified by immunostaining with MAP2ab and GFAP. Cells exposed to growth factor supplemented medium formed free-floating spheres within one week. Upon growth factor removal and plating on laminin-coated dishes, brain derived spheres gave rise to neurons, astrocytes and oJigodendrocytes; spinal cord derived spheres generated only astrocytes. This protocol describes an efficient method to generate and culture neurons and astrocytes from second trimester human CNS tissue that may be useful in transplantation. and developmental studies.

HIV infection of human fetal neural cells is mediated by gp120 binding to a cell membrane-associated molecule that is not CD4 nor galactocerebroside

HIV infection of central nervous system (CNS) tissue is a common finding in both adult and pediatric AIDS. Because most children are believed to be infected perinatally, we have developed a model of HIV CNS infection that utilizes explant organotypic cultures of human fetal CNS tissue. Using this model we previously reported that both lymphocytotropic and monocytotropic HIV isolates infect microglia and astrocytes. However, the mechanism by which HIV infects these cells remains to be elucidated. We have observed that neural cell infection in these cultures may be the result of receptor-mediated endocytosis. In order to confirm this observation and to determine the ligand responsible for this process, organotypic cultures were exposed to untreated HIV, HIV pretreated with soluble CD4 (sCD4) or, as a control, heat-inactivated HIV. To address the question of a putative receptor for HIV infection, CNS cultures were either untreated or pretreated with gp120 or with the deglycosylated form of this protein. Other cultures were treated with antibodies to CD4 (anti-T4A) or to galactocerebroside (GC). Results demonstrate that pretreatment of either HIV with sCD4 or CNS cultures with gp120 significantly inhibits HIV infection. The inhibition of infection was demonstrated by a reduction in the number of cells positive for HIV proteins and by decreases in HIV proviral DNA and p24 production. Pretreatment of CNS cultures with deglycosylated gp120, anti-T4A or anti-GC antibodies did not inhibit HIV infection. These data suggest that HIV gp120 is needed for binding to a surface molecule on CNS cells that is not CD4 nor GC and that this molecule may function as a receptor and lead to infection of neural cells.

Expression of Tumor Necrosis Factor α (TNFα) and Interleukin 6 (IL-6) mRNA in Adult Human Astrocytes

Astrocytes and microglia are cell populations which are implicated as being capable of regulating and effecting immune responses within the central nervous system (CNS). These functions are postulated to be mediated at least in part by production of soluble protein molecules termed cytokines. In this study, we utilized dissociated cultures of glial cells prepared from adult and fetal CNS tissue and a combined in situ hybridization-immunocytochemical technique in order to compare expression of TNF alpha and IL-6 mRNA between adult and fetal astrocytes and between adult astrocytes and microglia. Our results, using digoxygenin-labeled riboprobes, indicate that in contrast to fetal astrocytes only rare adult astrocytes express TNF alpha and IL-6 transcripts under our serum-supplemented basal culture conditions. Activation with LPS and IFN gamma increased the proportion of adult astrocytes expressing detectable TNF alpha and IL-6 mRNA signals; however, the proportion was significantly less than for microglia contained in the same cultures. These results suggest that microglia rather than astrocytes are more likely to be sources of these cytokines within the adult human CNS. Further studies of cytokine expression by glial cells will need to consider both the age and species of the glial cells used.

Angiogenesis and the blood-brain barrier in intracerebral solid and cell suspension grafts

Solid and suspension grafts of fetal central nervous system (CNS) tissue rapidly reform an intact blood-brain barrier (BBB), whereas solid grafts of peripheral nervous system (PNS) tissue fail to establish a BBB as detected by horseradish peroxide (HRP) leakage, administrated intravenously. We examined the acute changes in the BBB after grafting of fetal CNS tissue in solid and suspension form and superior cervical ganglion (SCG) and PNS tissue in the same manner. Adult rats (n = 20) received fetal (day 14–15) forebrain grafts (either solid or cell suspension) to their rostral corpus callosum bilaterally. A second group (n = 20) received SCG solid and cell suspension grafts at the same coordinates with the same technique. The animals were killed on first, third, seventh, and tenth days after grafting. Intravenous HRP (Sigma, type VI, 75 mg/5-g rat) was given 1 hour before perfusion with mixed aldehydes. Fifty-micron coronal sections were examined for the presence and location of the graft by cresyl violet and AChE staining and Mesulam's TMB method to detect HRP leakage. HRP leakage was detected in the parenchyma in all groups on the first and the third days post-transplantation indicating a disrupted BBB. No HRP reaction was seen at days 7 and 10 in groups receiving fetal forebrain tissue whether solid or cell suspension. Solid grafts of SCG consistently demonstrated HRP leakage from the first through the tenth day. However, cell suspension of SCG established a BBB by 7 days. These results suggest that within the solid grafts of CNS and PNS tissue, the permeability of the vessels is dictated by the transplanted tissue itself. When cell suspensions of the same tissue are introduced, host CNS tissue dominates as the local environment resulting in non-leaky vasculature within the graft.

Grafts of fetal central nervous system tissue rescue axotomized clarke's nucleus neurons in adult and neonatal operates

Many conditions are thought to contribute to neuron death after axotomy, including immaturity of the cell at the time of injury, inability to reestablish or maintain target contact, and dependence on trophic factors produced by targets. Exogenous application of neurotrophic factors and transplants of peripheral nerve and embryonic central nervous system (CNS) tissue temporarily rescue axotomized CNS neurons, but permanent rescue may require transplants that are normal targets of the injured neurons. We examined the requirements for survival of axotomized Clarke's nucleus (CN) neurons. Two months after hemisection of the spinal cord at the T8 segment, there was an ipsilateral 30% loss of neurons at the L1 segment in adult operates and a 40% loss in neonates. Transplants of embryonic spinal cord, cerebellum, and neocortex inserted into the T8 segment at the time of hemisection prevented virtually all of the cell death in both adults and neonates, but transplants of embryonic striatum were ineffective. None of the grafts prevented the somal atrophy of CN neurons caused by axotomy. Retrograde transport of fluoro-gold from the cerebellum demonstrated that 33% of all CN neurons at L1 project to the cerebellum, 50% of these died following a T8 hemisection, but all these projection neurons were rescued by a transplant of embryonic spinal cord. These results suggest that the rescue of axotomized CN neurons is relatively specific for the normal target areas of these neurons, but this specificity is not absolute and may depend on the distribution and synthesis of particular neurotrophic agents.

Scientific and ethical concerns in neural fetal tissue transplantation.

This report presents a brief overview of the medical and ethical issues involved with the procurement, preparation, safety, efficacy, and subject protection of human fetal central nervous system tissue in the context of neural transplantation. The ethical perspectives from which to view the clinical use of fetal tissue include the following: 1) that fetal tissue from elective abortions is a surgical specimen; 2) that the use of such tissue involves fetal experimentation in which the fetus is a subject; and 3) that fetal tissue is considered as a cadaveric organ specimen, similar to other organs, but with special considerations compared with adult cadaveric tissue. The latter approach appears to be the most applicable and is parallel to the use of cadaveric organs and tissues after a declaration of brain death. Additional issues include the following: 1) the safety and quality of fetal tissue for implantation; 2) the hypothesis that "legitimization" and "redemption" (potentially positive effects of tissue donation in general) may lead to an increase in elective abortion rates; 3) the ethical issues of the validity and value of human experimentation involving neural grafting; and 4) the type of consent to be obtained and the appropriate timing. Elective abortions, however, probably will continue to be the primary source of fetal tissue for grafting for some time, until other tissue sources become available.

Macrophage colony-stimulating factor in human fetal astrocytes and microglia. Differential regulation by cytokines and lipopolysaccharide, and modulation of class II MHC on microglia.

CSF-1 is a growth factor that selectively promotes the proliferation, survival, and differentiation of cells of the mononuclear phagocyte series. As part of a study on the role of cytokine and hematopoietic growth factors in central nervous system (CNS) development and inflammation, we examined the expression of CSF-1 in dissociated glial cells cultured from human fetal CNS tissue. CSF-1 mRNA and protein were constitutively expressed by astrocytes. The steady state level of CSF-1 mRNA was markedly up-regulated by both IL-1 beta and TNF-alpha in a time- and dose-dependent manner, whereas only a minimal increase was detected after stimulation with LPS. In unstimulated astrocyte cultures, CSF-1 protein levels gradually increased to 3.5-fold base-line values by 96 h and were significantly increased by all three stimulants in the order of IL-1 > or = TNF > LPS. Low levels of CSF-1 mRNA and protein were also detected in unstimulated microglia cultures. In contrast to astrocyte cultures, CSF-1 mRNA and protein increased significantly after stimulation with LPS, but changed only minimally after exposure to TNF-alpha or IL-1 beta. The effect of CSF-1 on cell proliferation, morphology, and class II MHC Ag expression was determined in highly enriched cultures of microglia and astrocytes. Microglia treated with CSF-1 showed a modest level of proliferation and differentiation into rod-shaped cells, whereas neither cell number nor shape was changed in astrocyte cultures. Interestingly, marked inhibition of both basal and IFN gamma-induced class II MHC Ag expression was observed in microglial cells cultured in the presence of CSF-1, whereas no effect was detected in astrocytes. These results suggest the possibility that in situ production of CSF-1 in the CNS may regulate normal glial cell development and contribute to the immunologic status of the CNS through the down-regulation of class II MHC expression.

In vivo magnetic resonance imaging of fetal cat neural tissue transplants in the adult cat spinal cord

Magnetic resonance (MR) imaging was evaluated for its possible diagnostic application in determining the survival of fetal central nervous system tissue grafts in the injured spinal cord. Hemisection cavities were made at the T11-L1 level of eight adult female cats. Immediately thereafter, several pieces of tissue, either obtained from the fetal cat brain stem on embryonic Day 37 (E-37), from the fetal neocortex on E-37, or from the fetal spinal cord on E-23, were implanted into the cavities made in seven cats. The eighth cat served as a control for the effect of the lesion only. In another group of four animals, a static-load compression injury was made at the L-2 level. Seven weeks later, the lesion was resected in three cases and fragments of either fetal brain-stem or spinal cord tissue were introduced. A small cyst was observed in a fourth cat in the compression injury group and a suspension of dissociated E-23 brain-stem cells was injected into this region of cavitation without disturbing the surrounding leptomeninges. Five months to 2 years posttransplantation, MR imaging was performed with a 2.0-tesla VIS imaging spectrometer by acquiring multislice spin-echo images (TR 1000 msec, TE 30 msec) in both the transverse and sagittal planes. Collectively, these intermediate-weighted images revealed homogeneous, slightly hyperintense signals at the graft site relative to the neighboring host tissue in seven of the 11 graft recipients. Two of the remaining four cats exhibited signals from the graft site that were approximately isointense with the adjacent host spinal cord, and the final two cats and the lesion-only control presented with very hypointense transplant/resection regions. The hyperintense and isointense images were tentatively interpreted as representing viable graft tissue, whereas the hypointense transplant/resection sites were considered to be indicative of a lack of transplant survival or the absence of tissue in the lesion-only control animal. Postmortem gross inspection of fixed specimens and light microscopy verified the MR findings in the control animal in 10 of the 11 graft recipients by showing either transplants and/or cysts corresponding to the MR images obtained. In one cat in the hemisection group, histological analysis revealed a very small piece of graft tissue that was not detected on the MR images. Therefore, it is suggested that within certain spatial- and contrast-resolution limits, MR imaging can reliably detect the presence of transplanted neural tissue in both the hemisected and compression-injured spinal cord of living animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional enhancement of intrastriatal dopamine-containing grafts by the co-transplantation of sciatic nerve tissue in 6-hydroxydopamine-lesioned rats

Peripheral nerve “bridges” demonstrate the ability to facilitate axonal growth and regenerate adult and fetal central nervous system tissue. The purpose of this study was to determine if co-grafted peripheral nerve tissue could enhance the ability of fetal dopamine (DA) cell transplants to reinnervate host striatum that had been denervated unilaterally. Male Fisher-344 rats were unilaterally lesioned with 6-hydroxydopamine to eliminate the nigrostriatal DA pathway. A total of 31 rats demonstrated a pattern of rotation indicative of a greater than 98% depletion in DA. Rats were kept as nongrafted controls ( n = 6), grafted with sciatic nerve (PN) minces ( n = 6), grafted with fetal ventral mesencephalon (VM; n = 10), or co-grafted with VM and PN minces ( n = 9). All groups were then tested for changes in apomorphine-induced rotational behavior. The PN control group showed no significant differences in rotation when compared to pregrafting levels and to the lesioned nongrafted group. Both the VM-grafted group and the VM-PN co-grafted group showed significant ( P < 0.01, one-way ANOVA) decreases in rotations beginning at 1.5 weeks postgrafting. There was a progressive decrease in rotations up to 12 weeks, the last test point examined. Interestingly, the co-graft group revealed a significantly greater decrease in rotation ( P < 0.05) than the VM group beginning at 5 weeks and continuing out to the 12-week test point. Histological and immunocytochemical studies showed good survival of both PN and VM grafts. The augmented recovery could not be accounted for by increased DA cell survival or host brain DA reinnervation in the co-graft group. Taken together, these findings suggest that PN tissue enhances the ability of fetal VM grafts to reinnervate host brain.

Detection of HIV in fetal central nervous system tissue

Neurological disease is a common finding in children with AIDS and in others without signs of disease but with evidence of congenital HIV-1 infection. To investigate the possibility that HIV-1 can infect fetal central nervous system (CNS) tissue and therefore possibly serve as the substrate for the abnormal neurodevelopment characteristic of pediatric AIDS, eight abortus CNS samples (one set of twins) from seven HIV-1-seropositive intravenous drug users (IVDUs) and eight control abortus CNS samples from eight HIV-1-seronegative IVDUs were analyzed for HIV-1 infection. HIV-1 nucleic acid was detected only after the use of polymerase chain reaction (PCR) in three of eight CNS samples from HIV-seropositive IVDUs but not in samples from seronegative subjects. In situ hybridization confirmed that HIV-1 DNA sequences were in cells in the CNS parenchyma of two of the three positive samples. This study demonstrates that HIV-1 can infect human fetal CNS tissue in vivo, but that the use of PCR may be necessary for its detection.

Localization of morphologically distinct microglial populations in the developing human fetal brain: implications for ontogeny

Although 70 years have elapsed since del Rio Hortega's initial description of microglia, the ontogeny of these cells remains enigmatic. In addition to the general scientific importance of clarifying this issue, a more complete characterization of microglia is dictated by their apparent pivotal role in the pathophysiology of central nervous system (CNS) disease associated with the acquired immunodeficiency syndrome (AIDS) in adults and, especially, in children. To accomplish this goal, fetal central nervous system tissue was collected at the time of elective pregnancy terminations. Coronal vibratome sections of Bouin's-fixed cerebrum were either stained with Ricinus communis agglutinin-1 (RCA-1) or analyzed by immunohistochemistry using monoclonal antibodies that recognize human tissue macrophages and microglia. By at least 13 weeks of gestation, microglia were detected in fetal brain. In the 13–18 week gestational age cerebrum, there was variability in the morphology of microglia within the developing white matter and cortex. However, there was less variability within these areas in the 19–24 week gestational age group. At all ages the greatest number of labeled cells per field was in the germinal matrix with decreasing numbers in the developing white matter and cortex. Cells in the germinal matrix were round with short processes (‘ameboid microglia’) while cortical cells were more ramified (‘resting microglia’). These results suggest that microglia may originate in the germinal matrix rather than in the pial mesenchyme as originally hypothesized by del Rio Hortega. Furthermore, because microglia may be the major cellular target for HIV-1 infection in the fetus, if they are not present in human fetal CNS prior to 13 weeks gestation then HIV-1 CNS infection would seem less likely prior to this age. Elucidation of these points might lead to improved preventative or therapeutic strategies in the treatment of perinatal and pediatric AIDS.

Collection and Use of Fetal Central Nervous System Tissue

A recent promising development in the field of central nervous system (CNS) tissue transplantation has suggested the use of human fetal CNS tissue from first trimester abortions for xenografting/explantation. Such experiments would certainly expand our knowledge of the normal developmental mechanisms in the human CNS, and allow studies of various indices of maturation and CNS function. However, the suggestion is looked upon with hesitance for ethical, legal and perhaps even for scientific reasons. The initial experiments have been very valuable, though, for our understanding of the structural and functional development of the human CNS, and several legal and ethical concerns have been addressed in working out the procedures for retrieving such tissue. This article tries to put our present knowledge in the right perspective of scientific achievements and potential, legal restrictions and ethical concerns.

Transplantation of fetal spinal cord tissue into the chronically injured adult rat spinal cord

Transplants of fetal central nervous system (CNS) tissue into the acutely injured rat spinal cord have been demonstrated to differentiate and partially integrate with the adjacent host neuropil. In the present study, we examined the potential for applying a transplantation approach to chronic spinal cord lesions. In particular, we were interested in learning whether host-graft fusion would be adversely affected by an advanced histopathology characterized in part by glial scar formation. Hemisection cavities were prepared at lumbar levels of the adult rat spinal cord 2-7 weeks prior to the transplantation of spinal cord tissue obtained from 14-day rat fetuses. Graft survival, differentiation, and integration with the host spinal cord were subsequently evaluated by light microscopic techniques at post-transplantation intervals of 1-6 months. Immunocytochemistry was also employed to examine the extent of astrocytic scar formation at the host-graft interface and serotoninergic innervation of the grafts. In some other cases, anterograde and retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase was used to determine whether axonal projections were formed between the host spinal cords and grafts. By 2 weeks after injury the initial lesion cavities were surrounded by a continuous astrocytic scar which remained intact for at least 7 weeks after injury in nongrafted control animals. In other animals, transplantation into these advanced lesions resulted in well-differentiated grafts with a 90% long-term survival rate. Although dense gliosis was still present along the lesion surfaces of the recipient spinal cord, foci of confluent host-graft neuropil were observed where interruptions in the scar had occurred. Donor tissue integrated most often with the host spinal cord at interfaces with host gray matter; however, some implants also exhibited sites of fusion with damaged host white matter. Thus, some regions of confluent graft and host neuropil could be routinely identified, despite the presence of a dense glial scar along the walls of the chronic lesion site at the time of transplantation. Anterograde and retrograde tract-tracing results suggested that some axonal projections into these grafts had originated from host neurons located immediately adjacent to the donor-recipient interface. In addition, immunocytochemistry revealed some host serotoninergic axons (presumably of supraspinal origin) traversing nongliotic interfaces. The results of this study raise the possibility that grafted fetal CNS tissue has a capacity for stimulating partial regression of an established glial scar.(ABSTRACT TRUNCATED AT 400 WORDS)

Collection and use of aborted central nervous system material. Commendation and controversy.

A recent promising development in the field of central nervous system (CNS) transplantation has suggested the use of human fetal CNS tissue from first trimester abortions for xenografting/explantation. Such experiments would certainly expand our knowledge of the normal developmental mechanisms in human CNS, and allow studies of various indices of maturation and CNS function. However, the suggestion is looked upon with hesitance for ethical, legal and perhaps even for scientific reasons. The initial experiments have been very valuable, though, for our understanding of the structural and functional development of the human CNS, and several legal and ethical concerns have been addressed in working out the procedures for retrieving such tissue. This article tries to put our present knowledge in the right perspective of scientific achievements and potential, legal restrictions and ethical concerns.