By 2050, the number of people age 65 and older with Alzheimer’s disease may nearly triple to a projected 13.8 million, with one new case appearing every 33 seconds.1 Neurodegeneration causes problems with movement and/or cogni-tive function, and includes a number of diseases such as Alzheimer’s, Parkinson’s, Multiple Sclerosis and Amyotrophic Lateral Sclerosis.2 The majority of neurodegenerative diseases are incurable, debilitating conditions that result in the progressive degeneration and/or death of nerve cells.2

Cell Culture- 1. AHPCs isolated from adult Fischer 344 rats and retrovirally infect-ed to express green �uorescent protein (GFP) were cultured in po-ly-orinthine laminin �asks 2. Cells were detached from the �ask, harvested by centrifugation, and plated at an approximate density of 10,000 cells per coverslip. 3. AHPCs were kept in maintenance media for 1 day. The next day, cells were placed in di�erentiation medium for 5 days in vitro (DIV) at an experimental temperature of 34°C and a control of 37°C.

Stem cell technologies have become increasingly attractive options to investigate and treat neurodegenerative diseases.2 Stem cells are undif-ferentiated cells found in multicellular organisms that are capable of self-renewal and di�erentiation into other cell types.2 By using targeted di�erentiation, developing more e�cient methods of isolating stem cells, and re�ning cell transplantation techniques, there is potential for future therapeutic applications to treat neurodegenerative disease.2

Adult hippocampal progenitor cells (AHPCs) are multipotent stem cells located in the brain with limited di�erentiation potential.3 They can give rise to the three major cell types of the central nervous system, neurons, astrocytes, and oligodendrocytes.3 Their di�erentiation is regulated by

In order to test the e�ect of external factors on AHPC di�erentiation we plan to transplant rat AHPCs into zebra�sh em-bryos. Zebra�sh are a good model because they are cost e�ective, can produce many embryos at a time, are transpar-ent for the �rst 24 hours, and develop a nervous system within 48 hours.4 Bastula stage zebra�sh embryos are an envi-ronment in which maximum cell plasticity may be observed. A potential limitation of this model is that zebra�sh devel-opment is optimal at 29°C, while AHPC di�erentiation is optimal at 37°C.5 This experiment will determine whether the di�erentiation of AHPCs and the development of zebra�sh varies at a median temperature of 34°C.

Fig 1. The regulation of self-renewal and di�erentiation of neural stem cells includes internal and external factors.6

Data suggests that the experimental temperature, at these three time points, may...

1. Not a�ect the di�erentiation of AHPCs into astrocytes. GFAP immunoreactivity was consistent across groups. 2. May reduce the amount of proliferation. Ki67 immunoreactivity was lower in the treatment group. 3. Likely does not a�ect the rate of AHPC di�erentiation. Nestin values are consistent across groups. 4. Does not a�ect the survival of zebra�sh, but we lack data for the �rst 24 hours. 5. Negatively a�ects zebra�sh length at 1 dpf, but no di�erence is observed at later time points.

In replicate experiments working with AHPCs we would plate the cells at a lower density, hoping that this will yield lower cell con�uence, decreased formation of nuerospheres, and fewer cells washing away during the immunocytochemistry process. Greater care will also be taken to avoid areas of high cell density and auto-�uorescence during cell imaging (as that made collecting reliable data di�cult). In replicate zebra�sh experiments we would use other metrics to measure �sh development, like acetylated tubulin staining for axons and swim bladder size, while also increasing the sample size.

Xenotransplatation of mammalian cells using a zebra�sh model has promising applications for future study of stem cells and cancer. The Sakaguchi Lab at Iowa State University is working to develop a functional model for transplantation. To use this technology we must �rst understand how varying temperature might a�ect the di�erentiation of transplanted cells, and the microenvironment in which they are transplanted. Our results indicate that temperature is a factor that should be considered in zebra�sh xenotransplantation models.

Treatment Temperature 34°C Control Temperature 37°C

Working with AHPCs Working with Zebra�sh

Imaging- 7. Imaging was done using a �uorescence microscope (Nikon Micro-phot FXA, Tokyo, Japan), equipped with a Retiga 2000R digital camera. 8. Images were captured using QCapture software, colorized with Photoshop, and analyzed using ImageJ.

3. Methods

Immunocytochemistry- 4. Fixation - Cells were �xed at 1, 3, and 5 days by placing them in a 4% Paraformaldehyde solution. 5. Labeling - Cells were immunolabeled with the primary antibodies summarized in Figure 2. 6. Cells were then immunolabeled with a secondary antibody carry-ing a red �uorescent marker.

Imaging- 5. Zebra�sh were imaged using a stereo-scope and ImageJ was used to determine �sh length.

Fixation- 4. Embryos were collected 1, 3, and 5 days post fertilization (dpf ) and their tissue was �xed in paraformaldehyde for 3 hrs.

Incubation- 3. The zebra�sh were divided into two groups and incubated on agarose plates with a penicillin/streptomycin antibiotic solution, at an experimental temperature of 34°C and a control temperature of 29°C.

Dechorionization- 2. 3 hours after fertilization the embryos were placed in a trypsin solution to remove the protective covering called the chorion.

Harvesting- 1. Tg(Flk:mCherry) zebra�sh were bred to produce embryos for the experiment.

1. Introduction

Fig 3. Images labeled A correspond with positive staining for the antibody noted in the top right corner. Images labled B show DAPI staining for all nuclei pres-ent. Images labeled C show a merged image of A and B, as well as GFP expressed by our transgenic AHPC cells.

Fig 2. Description of primary antibodies used, the cell structure it binds to, the cell type it stains, and the dilution used in the experiment.

4. Results

2. Experimental Questions 1. Is AHPC di�erentiation di�erent at a temperature of 34°C rather than their optimal temperature of 37°C? 2. Do zebra�sh develop di�erently at 34°C rather than their optimal temperature of 29°C?

5. Discussion

Fig 4. Graphs depict the average precentage of DAPI stained immunoreactive cells for each anti-body, at each time point. For each treatment at each timepoint we imaged 10 sites and counted the number of positive cells. For the days labeled with an asterisk less then 10 usable sites were present. A cell was counted as positive based on its brightness and staining pattern.

Fig 5. (A) Graph depicts survival of zebra�sh at each time point. There is no signi�cant di�erence be-tween treament groups at each time point. (B) Graph shows average zebra�sh length at each time point. There is a signi�cant di�erence between lengths at1dpf, but not at other times. A one way ANOVA was performed to determine signi�cance where **** p<0.0001.

Fig 6. Compares morphology of zebra�sh at each time point. Besides length at 1 dpf no physical di�er-ences were apparent.

internal and external factors and can be characterized by the di�erent proteins they synthesize.3

Acknowledgements

References

2. Gage, F. H. (2000). Mammalian neural stem cells. Science, 287(5457), 1433-1438.

1. Alzheimer's Association. (2011). 2011 Alzheimer's disease facts and �g-ures.Alzheimer's & dementia: the journal of the Alzheimer's Association, 7(2),

4. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., & Schilling, T. F. (1995). Stages of embryonic development of the zebra�sh. Developmental dynamics, 203(3), 253-310.

3. Palmer, T. D., Takahashi, J., & Gage, F. H. (1997). The adult rat hippocampus contains primordial neural stem cells. Molecular and Cellular Neuroscience, 8(6), 389-404.

5. Vittori, M., Motaln, H., & Turnšek, T. L. (2015). The study of glioma by xeno-transplantation in zebra�sh early life stages. Journal of Histochemistry & Cyto-chemistry, 0022155415595670.

6. Han, S., Yang, K., Shin, Y., Lee, J. S., Kamm, R. D., Chung, S., & Cho, S. W. (2012). Three-dimensional extracellular matrix-mediated neural stem cell di�erentia-tion in a micro�uidic device. Lab on a Chip, 12(13), 2305-2308.

Stem Cell Research Fund

Thank you to Adah Leshem, Stacy Renfro, Jennifer Lillo and all the CBiRC Sta�. RET Lead Teachers Craig Walter, Maureen Gri�n, and Eric Hall. Cells were donated by Fred Gage, PhD. of the Salk Institute. Special thanks to Bhavika, Grace, Emily, Marissa, Indy and all the members of Sakaguchi Lab.

Alex Conyers, Mai Dang, Elizabeth Sandquist, Je�rey Essner and Donald SakaguchiDepartment of Genetics, Development, and Cell Biology1210 Mol-Bio, 2437 Pammel Dr, Ames, IA 50011-1079

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