Global gene and cell replacement strategies via stem cells.

The inherent biology of neural stem cells (NSCs) endows them with capabilities that not only circumvent many of the limitations of other gene transfer vehicles, but that enable a variety of novel therapeutic strategies heretofore regarded as beyond the purview of neural transplantation. Most neurodegenerative diseases are characterized not by discrete, focal abnormalities but rather by extensive, multifocal, or even global neuropathology. Such widely disseminated lesions have not conventionally been regarded as amenable to neural transplantation. However, the ability of NSCs to engraft diffusely and become integral members of structures throughout the host CNS, while also expressing therapeutic molecules, may permit these cells to address that challenge. Intriguingly, while NSCs can be readily engineered to express specified foreign genes, other intrinsic factors appear to emanate spontaneously from NSCs and, in the context of reciprocal donor-host signaling, seem to be capable of neuroprotective and/or neuroregenerative functions. Stem cells additionally have the appealing ability to 'home in' on pathology, even over great distances. Such observations help to advance the idea that NSCs - as a prototype for stem cells from other solid organs - might aid in reconstructing the molecular and cellular milieu of maldeveloped or damaged organs.

Ectopic expression of the neural cell adhesion molecule L1 in astrocytes leads to changes in the development of the corticospinal tract.

The cell recognition molecule L1, of the immunoglobulin superfamily, participates in the formation of the nervous system and has been shown to enhance cell migration and neurite outgrowth in vitro. To test whether ectopic expression of L1 would influence axonal outgrowth in vivo, we studied the development of the corticospinal tract in transgenic mice expressing L1 in astrocytes under the control of the GFAP-promoter. Corticospinal axons innervate their targets by extending collateral branches interstitially along the axon shaft following a precise spatio-temporal pattern. Using DiI as an anterograde tracer, we found that in the transgenic animals, corticospinal axons appear to be defasciculated, reach their targets sooner and form collateral branches innervating the basilar pons at earlier developmental stages and more diffusely than in wild type littermates. Collateral branches in the transgenic mice did not start out as distinct rostral and caudal sets, but they branched from the axon segments in a continuous rostrocaudal direction across the entire region of the corticospinal tract overlying the basilar pons. The ectopic branches are transient and no longer present at postnatal day 22. The earlier outgrowth and altered branching pattern of corticospinal axons in the transgenics is accompanied by an earlier differentiation of astrocytes. Taken together, our observations provide evidence that the ectopic expression of L1 on astrocytes causes an earlier differentiation of these cells, results in faster progression of corticospinal axons and influences the branching pattern of corticospinal axons innervating the basilar pons.

Segregation of human neural stem cells in the developing primate forebrain.

Many central nervous system regions at all stages of life contain neural stem cells (NSCs). We explored how these disparate NSC pools might emerge. A traceable clone of human NSCs was implanted intraventricularly to allow its integration into cerebral germinal zones of Old World monkey fetuses. The NSCs distributed into two subpopulations: One contributed to corticogenesis by migrating along radial glia to temporally appropriate layers of the cortical plate and differentiating into lamina-appropriate neurons or glia; the other remained undifferentiated and contributed to a secondary germinal zone (the subventricular zone) with occasional members interspersed throughout brain parenchyma. An early neurogenetic program allocates the progeny of NSCs either immediately for organogenesis or to undifferentiated pools for later use in the "postdevelopmental" brain.

Neural stem cells are uniquely suited for cell replacement and gene therapy in the CNS.

In recent years, it has become evident that the developing and even the adult mammalian CNS contain a population of undifferentiated, multipotent cell precursors, neural stem cells, the plastic properties of which might be of advantage for the design of more effective therapies for many neurological diseases. This article reviews the recent progress in establishing rodent and human clonal neural stem cell lines, their biological properties, and how these cells can be utilized to correct a variety of defects, with prospects for the near future to harness their behaviour for neural stem cell-based treatment of diseases in humans.

Neural stem cells -- a versatile tool for cell replacement and gene therapy in the central nervous system.

In recent years, it has become evident that the developing and even the adult mammalian central nervous system contains a population of undifferentiated, multipotent cell precursors, neural stem cells, the plastic properties of which might be of advantage for the design of more effective therapies for many neurological diseases. This article reviews the recent progress in establishing rodent and human clonal neural stem cell lines, their biological properties, and how these cells can be utilized to a correct variety of defects, with prospects for the near future to harness their behaviour for neural stem cell-based treatment of diseases in humans.

Remodeling of lesioned kitten visual cortex after xenotransplantation of fetal mouse neopallium.

Remodeling of the mechanically injured cerebral cortex of kittens was studied in the presence of a neural xenograft taken from mouse fetuses. Solid neural tissue from the neopallium of a 14-day-old fetus was transferred into a cavity prepared in visual cortical area 18 of 33-day-old kittens. Injections of bromodeoxyuridine (BrdU) were used to monitor postoperative cell proliferation. Two months after transplantation, the presence of graft tissue in the recipient brain was assessed by Thy-1 immunohistochemistry. Antibodies specific for neurons, astrocytes, and oligodendrocytes and hematoxylin staining for endothelial cells were used for the characterization of proliferating (BrdU+) cells. The following were the major observations: 1) Of ten transplanted kittens, four had the cavity completely filled with neural tissue that resembled the intact cerebral cortex in its cytoarchitecture, whereas, in four other kittens, the cavity was partially closed. In two kittens, the cavity remained or became larger, which was also the case with all four sham-operated (lesioned, without graft) animals. 2) A substantial part of the remodeled tissue was of host origin. Only a few donor cells survived and dispersed widely in the host parenchyme. 3) In the remodeled region of transplanted animals, the densities of nerve, glial, and endothelial cells were similar to those in intact animals. 4) Cell proliferation increased after transplantation but only within a limited time, because, 2 months after the operation, the number of mitotic cells in the grafted cerebral cortex did not differ from that in intact controls. Our data suggest that the xenograft evokes repair processes in the kitten visual cortex that lead to structural recovery from a mechanical insult. The regeneration seems to rely on a complex interplay of many different mechanisms, including attenuation of necrosis, cell proliferation, and immigration of host cells into the wounded area.

Preservation of the structural integrity of a freshly lesioned or transplanted mouse neocortex and the immunoreactivity of cell-specific marker proteins in demineralized histological material.

Analysis of superficially lesioned or grafted brains poses the problem that their removal from the skull prior to histological processing often causes damage to the operated area and may lead to loss of the graft. Here, we propose an original approach to this problem, developed on mice whose cortices have been surgically lesioned and some grafted with fetal neural tissue. The experimental animals were killed 1, 3 or 6 days after operation. Our procedure was based on the softening of skulls by demineralization in Gendre's picric solution, followed by solidification of the wounded region by embedding in polyester wax. This permitted the preparation of serial sections from brains together with neurocrania. To check their immunoreactivity, the sections were later reacted with specific antisera for glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), calbindin, and the thermolabile cell-surface glycoprotein Thy-1. The histological material revealed excellent structural integrity and cytoarchitecture. In transplanted animals, the tiny graft, protected by the overlying bone, was found in the host cavity. Immunostaining showed typical localization of the chosen marker proteins. The anti-Thy-1 antibody enabled us to distinguish between graft and host tissues, which differed, in our experiments, in their expression of two distinct allelic forms of the Thy-1 molecule. The method lends itself perfectly to histochemical study of the earliest stages of freshly operated superficial brain regions in small laboratory animals, and should also be applicable to the evaluation of other brain structures which are difficult to gain access to without being damaged.

Newly formed host cells in a grafted juvenile neocortex express neurone-specific marker proteins.

Recently we reported that a mouse foetal neural graft, when transferred into a lesioned juvenile neocortex, may induce cortical repair and stimulate proliferation of the host cells. The present study was focused on an immunohistochemical identification of neurones among these newly generated cells. Adjacent sections from the brains already used in our previous study were stained either with an antibody against the host-specific Thy-1 antigen, or with neurone-specific antibodies recognizing the microtubule-associated protein MAP2, the heavy subunit of neurofilaments and parvalbumin. Dividing cells, labelled repeatedly during the first three post-operative days with 3H-thymidine, were detected after 2 months by autoradiography. We found that in the repaired neocortical region newly formed host cells, whose distribution resembled the one found in an intact neocortex, also contained neurones. These new data corroborate our previous suggestion that a juvenile mammalian neocortex participates, after lesioning and under the presence of a foetal neural graft, in its own repair by the formation of new cells, including neurones.

Do foetal neural grafts induce repair by the injured juvenile neocortex?

Repair of mechanically injured primary somatosensory cortex in 3 week old mice was studied by placing small, solid foetal neurotransplants into large cortical cavities. After transplantation, the graft and host tissues were distinguished immunocytochemically owing to their expression of two different Thy-1 antigens. Cell proliferation was monitored by 3H-thymidine autoradiography. The following observations were made two months after operation: (i) In 8 out of 11 grafted animals new cortical tissue had taken the place of the cavity. (ii) Five of these 8 animals contained only host tissue; the remainder presented a small piece of grafted tissue. (iii) In the restored cortical area, newly generated cells were predominantly of host origin. These data suggest that the restorative capacity of the already post-mitotic cerebral cortex is not lost and may be reactivated. The presence of a foetal neural graft seems to favour this process.

Positive feedback effect of dihydrotestosterone on follicle-stimulating hormone secretion in the male rat: implications and a possible relation to the onset of puberty.

Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were measured in serum of adult male rats after 6 days of constant subcutaneous infusion of varying levels of dihydrotestosterone (DHT). Doses from one-half up to the normal "blood production rate" of DHT produced a selective stimulation of serum FSH, but not LH, levels. Higher levels suppressed FSH, LH, and testosterone. Despite the presence of much higher levels of testosterone in blood, the augmentation of FSH secretion indicated in these studies suggests that DHT may have an important role in regulatory systems for gonadotropins.

Blunted prolactin response to hypoglycemia in patients with hypothalamic-pituitary disease and in subjects receiving estrogen.

The prolactin response to hypoglycemia was evaluated in 22 control subjects and 8 patients with hypothalamic-pituitary disease but normal basal serum prolactin levels. Eighteen of the 22 control subjects demonstrated at least a twofold prolactin rise in response to hypoglycemia. In contrast to the control subjects, none of the 8 patients demonstrated a prolactin response to hypoglycemia. This blunted prolactin response to hypoglycemia was the only endocrine abnormality in 3 of these 8 patients. In an attempt to better determine the sensitivity of the prolactin response to hypoglycemia as an index of early pituitary disease, the effect of a short course of estrogen on the prolactin response to hypoglycemia was examined. Estrogen was selected because of its known acute stimulatory effect on pituitary mitosis and chronic effects that lead to pituitary tumor formation in rodents. Accordingly, diethylstilbestrol (DES) 5 mg t.i.d. was administered orally to 6 normal men for 3 days, a period known to stimulate pituitary mitotic activity in rodents. Diethylstilbestrol treatment caused significant elevation of the baseline prolactin (8 +/- 2 versus 18 +/- 3 ng/ml, p less than 0.05); however, the prolactin response to hypoglycemia was blunted (8 +/- 2--30 +/- 10 ng/ml, p less than 0.05, before DES; 18 +/- 3--20 +/- 5 ng/ml after DES, p greater than 0.05). This estrogen-induced blunted prolactin response to hypoglycemia resembled the blunted prolactin response to hypoglycemia found in patients with hypothalamic-pituitary disease.

PIP: This study investigated the prolactin response to hypoglycemia in patients with a variety of diseases of the hypothalamic--pituitary axis but with normal basal serum prolactin levels and in normal subjects who received a short course of estrogen, administration of which is known to stimulate prolactin secretion and pituitary mitotic activity in experimental animals, which suggests that estrogen-induced hyperprolactinemia might mark pituitary hyperplasia. Hence, the prolactin response to hypoglycemia was evaluated in 22 controls and 8 patients. 18 of the 22 controls showed at least a 2-fold prolactin rise in response to hypoglycemia. Compared to controls, 0 of 8 patients demonstrated prolactin response to hypoglycemia. To test whether the sensitivity of this prolactin response to hypoglycemia could be an index of early pituitary disease, the short course of estrogen was administered (diethylstilbestrol, 5 mg, t.i.d.) orally to 6 normal men for 3 days. Diethylstilbestrol treatment resulted in significant elevation of baseline prolactin (p .05); On the other hand, prolactin response to hypoglycemia was blunted (P .05 before estrogen and P .05 after estrogen). The blunted response induced by estrogen administration was similar to that seen in patients with hypothalamic-pituitary disease in response to hypoglycemia.