A rapid and sensitive method for detecting human cells in xenogeneic hosts.

Xenografts, specifically transplantation of human cells into other species, are a valuable tool in preclinical transplantation experiments. A central issue is accurate identification of the grafted cells, particularly in cases in which cellular migration has occurred. We report that detection of grafted human cells can be achieved by in situ hybridization techniques using human centromeric probes which result in unambiguous nuclear labeling. The resulting reaction can be combined with immunocytochemical or histochemical techniques for cell-type characterization.

Transplant therapy: recovery of function after spinal cord injury.

Spinal cord injuries (SCI) result in devastating loss of function and altered sensation. Presently, victims of SCI have few remedies for the loss of motor function and the altered sensation often experienced subsequent to the injury. A goal in SCI research is to improve function in both acute and chronic injuries. Among the most successful interventions is the utilization of transplanted tissues toward improved recovery. The theory is that the transplanted tissue could (1) bridge the spinal lesion and provide chemical and/or mechanical guidance for host neurons to grow across the lesion, (2) bridge the spinal lesion and provide additional cellular elements to repair the damaged circuitry, (3) provide factors that would rescue neurons that would otherwise die and/or modulate neural circuits to improve function. A variety of tissues and cells have been added to the adult mammalian spinal cord to encourage restoration of function. These include Schwann cells, motor neurons, dorsal root ganglia, adrenal tissue, hybridomas, peripheral nerves, and fetal spinal cord (FSC) tissue en bloc or as disassociated cells. It is postulated that these tissues would rescue or replace injured adult neurons, which would then integrate or promote the regeneration of the spinal cord circuitry and restore function. In some instances, host-appropriate circuitry is supplied by the transplant and functional improvement is demonstrated. In this presentation, specific examples of recent work with transplanted tissue and cells that demonstrate improved behavioral outcome are presented. New recent work describing the in vitro propagation and characterization of human fetal spinal cord multipotential progenitor cells are also described in the context of a potential resource for transplantable cells. Additionally, data from transplantation experiments of human FSC cells into nonimmunosuppressed rat spinal cord are described, and the resultant improvements in behavioral outcome reported. Lastly, directions for future SCI research are proposed.

Migration and differentiation of PC12 cells transplanted into the rat spinal cord.

To test the hypothesis that transplanted neuronal or neuronal-like cell lines, grown in vitro, might survive and differentiate in the mammalian spinal grey matter, adult male Sprague-Dawley rats (N = 5) were injected with a suspension of between 3 x 10(5) and 1.0 x 10(6) DiI labeled, undifferentiated rat pheochromocytoma (PC12) cells in sterile phosphate buffered saline. The PC12 cell line was chosen since, in certain in vitro conditions, this cell line serves as a model of neuronal differentiation, which includes the ability to conduct action potentials and form functional synapses. After a survival time of 7 or 8 days, the spinal cords were removed, cryosectioned longitudinally and examined for detection of DiI labeled PC12 cells using fluorescent microscopy. The number of DiI labeled profiles and the proportions of the DiI cells which were differentiated were counted per section in at least five non-contiguous sections per animal. Differentiation was defined as cells with neurite-like extension which exceeded twice the soma diameter. Results demonstrated the following: (1) from 2 to 15% of the transplanted PC12 cells survived; (2) migration within the spinal grey matter occurred since PC12 cells were found as much as 510 microns away from the injection site; (3) of the surviving PC12 cell population, a proportion of between 60 and 80% were differentiated, many with two or more neurite-like processes, in all of the rats; (4) no mitotic profiles were observed in DiI labelled cells; (5) undifferentiated PC12 cells were juxtaposed to the lumens of small blood vessels or within the lesion cavity. Although the specific factors remain to be elucidated, the observed PC12 migration and differentiation within the host spinal grey matter appears to be controlled by factors in the microenvironment. These data support the use of a homogeneous in vitro population of neuronal or neuronal-like cells, which are readily accessible to transfection with the appropriate genes, as transplant sources for the injured spinal cord.

Early detection of colorectal cancer by quantitative fluorescence image analysis of exfoliated cells.

Early-stage colorectal cancer is potentially curable. In the present study, we applied quantitative fluorescence image analysis (QFIA) cytology to the detection of experimental colorectal cancer in a rodent model. QFIA cytology combines visual cytologic examination with quantitation of DNA content in single exfoliated cells. Cancer was induced by treating 110 rats with subcutaneous 1,2-dimethylhydrazine. Sequential colon washes were obtained weekly from each animal for 20 weeks. Control animals were treated identically except for the administration of carcinogen. Cells that were cytologically abnormal or had increased DNA content were found starting in the second week. By the eighth week, roughly 50 percent of animals had positive results, and this level remained approximately constant for the duration of the study. Tissue pathologic results were normal during weeks 1 to 7. Dysplasias became common during weeks 8 to 15 whereas most cancers appeared during weeks 16 to 21. These results indicate that QFIA cytology is a highly sensitive method for detecting even preneoplastic changes resulting from carcinogen administration and may prove useful in detecting human colorectal cancer.