Structural recovery in lesioned adult mammalian spinal cord by x-irradiation of the lesion site.

Mechanical injury to the adult mammalian spinal cord results in permanent morphological disintegration including severance/laceration of brain-cord axons at the lesion site. We report here that some of the structural consequences of injury can be averted by altering the cellular components of the lesion site with x-irradiation. We observed that localized irradiation of the unilaterally transected adult rat spinal cord when delivered during a defined time-window (third week) postinjury prevented cavitation, enabled establishment of structural integrity, and resulted in regrowth of severed corticospinal axons through the lesion site and into the distal stump. In addition, we examined the natural course of degeneration and cavitation at the site of lesion with time after injury, noting that through the third week postinjury recovery processes are in progress and only at the fourth week do the destructive processes take over. Our data suggest that the adult mammalian spinal cord has innate mechanisms required for recovery from injury and that timed intervention in certain cellular events by x-irradiation prevents the onset of degeneration and thus enables structural regenerative processes to proceed unhindered. We postulate that a radiation-sensitive subgroup of cells triggers the delayed degenerative processes. The identity of these intrusive cells and the mechanisms for triggering tissue degeneration are still unknown.

Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

Whole-body protochordate regeneration from totipotent blood cells.

Cell differentiation, tissue formation, and organogenesis are fundamental patterns during the development of multicellular animals from the dividing cells of fertilized eggs. Hence, the complete morphogenesis of any developing organism of the animal kingdom is based on a complex series of interactions that is always associated with the development of a blastula, a one-layered hollow sphere. Here we document an alternative pathway of differentiation, organogenesis, and morphogenesis occurring in an adult protochordate colonial organism. In this system, any minute fragment of peripheral blood vessel containing a limited number of blood cells isolated from Botrylloides, a colonial sea squirt, has the potential to give rise to a fully functional organism possessing all three embryonic layers. Regeneration probably results from a small number of totipotent stem cells circulating in the blood system. The developmental process starts from disorganized, chaotic masses of blood cells. At first an opaque cell mass is formed. Through intensive cell divisions, a hollow, blastula-like structure results, which may produce a whole organism within a short period of a week. This regenerative power of the protochordates may be compared with some of the characteristics associated with the formation of mammalian embryonal carcinomous bodies. It may also serve as an in vivo model system for studying morphogenesis and differentiation by shedding more light on the controversy of the "stem cell" vs. the "dedifferentiation" theories of regeneration and pattern formation.

Platelet-derived growth factor BB induces functional vascular anastomoses in vivo.

Neovascularization that generates collateral blood flow can limit the extent of tissue damage after acute ischemia caused by occlusion of the primary blood supply. The neovascular response stimulated by the BB homodimeric form of recombinant platelet-derived growth factor (PDGF-BB) was evaluated for its capacity to protect tissue from necrosis in a rat skin flap model of acutely induced ischemia. Complete survival of the tissue ensued, when the original nutritive blood supply was occluded, as early as 5 days after local PDGF-BB application, and the presence of a patent vasculature was evident compared to control flaps. To further evaluate the vascular regenerative response, PDGF-BB was injected into the muscle/connective tissue bed between the separated ends of a divided femoral artery in rats. A patent new vessel that functionally reconnected the ends of the divided artery within the original 3- to 4-mm gap was regenerated 3 weeks later in all PDGF-BB-treated limbs. In contrast, none of the paired control limbs, which received vehicle with an inactive variant of PDGF-BB, had vessel regrowth (P < 0.001). The absence of a sustained inflammatory response and granulation tissue suggests locally delivered PDGF-BB may directly stimulate the angiogenic phenotype in endothelial cells. These findings indicate that PDGF-BB can generate functional new blood vessels and nonsurgically anastomose severed vessels in vivo. This study supports the possibility of a therapeutic modality for the salvage of ischemic tissue through exogenous cytokine-induced vascular reconnection.

Adenovirus-mediated urokinase gene transfer induces liver regeneration and allows for efficient retrovirus transduction of hepatocytes in vivo.

Retrovirus-mediated gene transfer into hepatocytes in vivo results in long-term gene expression. Limitations include the need to remove two-thirds of the liver and the relatively low frequency of gene transfer. To increase gene transfer without surgical hepatectomy, mouse hepatocytes were transduced in vivo with a recombinant adenovirus that transiently expressed urokinase, resulting in high rates of asynchronous liver regeneration. During the regenerative phase, in vivo retroviral-mediated gene transfer in hepatocytes resulted in 5- to 10-fold greater transduction efficiencies than that obtained by conventional partial hepatectomy. In 3-4 weeks, the architecture and microscopic structure of the recipient livers were normal. The two-viral system of achieving permanent transgene expression from hepatocytes in vivo offers an alternative approach to current ex vivo and in vivo gene-transfer models.

Induction of cell proliferation in mammalian inner-ear sensory epithelia by transforming growth factor alpha and epidermal growth factor.

Regenerative proliferation occurs in the inner-ear sensory epithelial of warm-blooded vertebrates after insult. To determine how this proliferation is controlled in the mature mammalian inner ear, several growth factors were tested for effects on progenitor-cell division in cultured mouse vestibular sensory epithelia. Cell proliferation was induced in the sensory epithelium by transforming growth factor alpha (TGF-alpha) in a dose-dependent manner. Proliferation was also induced by epidermal growth factor (EGF) when supplemented with insulin, but not EGF alone. These observations suggest that stimulation of the EGF receptors by TGF-alpha binding, or EGF (plus insulin) binding, stimulates cell proliferation in the mature mammalian vestibular sensory epithelium.