Summary

בידוד הרחבת מבשרי עצבית בהיפוקמפוס כלב מבוגר

Published: November 29, 2016
doi:

Summary

המוח הכלבי הוא מודל ערך שבו ללמוד neurogenesis למבוגרים. מתפרסמים כאן פרוטוקולים לבידוד והרחבת תאים עצביים מבשרים כלבי מבוגרים בהיפוקמפוס מרקמות המוח עיקריות.

Abstract

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

Introduction

Regional variations in the rate of neurogenesis have been observed along the dorsoventral axis of the rodent hippocampus1,2. Furthermore, the rates of hippocampal neurogenesis also show distinct inter-species variation, with precursor cell turnover in the subgranular zone shown to be significantly lower in adult humans than in rodents3-5. Inter-species differences in hippocampal structural anatomy may be relevant here, as it has been postulated that neural stem cell distribution along the murine ventricular neuraxis may be influenced by cephalic flexures during embryological development6. To date, the rodent brain remains the most popular system in which to study adult neurogenesis. However, the brain of the domestic dog (Canis familiaris), with a size and structural organization intermediate between that of humans and rodents7, represents a valuable yet highly underused animal model. The canine hippocampus in particular embodies this structurally intermediate nature8-10 and can provide a unique perspective on intrinsic variations in neural precursor cell populations. With many closer parallels to the human brain, the canine model may also offer insight into the biology of adult human neurogenesis.

In vitro culture assays have become a key tool for the study of neural precursors and the cellular and biomolecular processes of adult neurogenesis. The neurosphere assay and adherent monolayer culture represent the two predominant systems for expanding neural precursor cells in vitro11-13. Protocols for brain extraction, hippocampal microdissection or neural precursor culture assays have been well documented for the rodent model14-16. However, for the adult canine brain they remain comparatively few17,18, focused instead on fetal or neonatal tissue19-21.

In our published study7 we investigated regional variations in neurogenesis and neural precursor cell populations across the dorsoventral axis of the adult canine hippocampus. Although highly dependent on breed, adulthood in canines is reached between 1 and 3 years of age. Here, we present detailed methods for the extraction, isolation and culture of neural precursor cells from the canine hippocampus. We provide systematic protocols for the expansion of neural precursor cells as both floating neurospheres and as an adherent monolayer culture, and for their subsequent differentiation into mature neural cell types.

Protocol

בהתאם ניו סאות 'ויילס, אוסטרליה החוק, רקמת המוח פוסט מורטם נרכשה כלבים בוגרים מורדמים מסיבות שאינן קשורות למחקר. 1. הכנת בינוני תרבות הכן פתרון ג'לטין 0.1% על ידי הוספת 0.1 גרם של אבקת ג&#39…

Representative Results

באמצעות שימוש מבחני מבשר במבחנה עצביים, הנוירוגנזה אוכלוסיות תאים עצביים מבשר התאפיינו ולעומת פני ציר dorsoventral של ההיפוקמפוס כלבים בוגרים. תאים עצביים מבשרים נגזרים רקמות בהיפוקמפוס מבודדות נוצר צף neurospheres בתוך 14 ימים של בידוד, להגיע בקוטר של 100 …

Discussion

הפרוטוקולים המתוארים כאן מותאמים לשמור על תנאי תרבות נוחים למקסם כדאי תא. המהירות טופל במהלך חילוץ, בידוד והרחבה היא בעלת חשיבות מכרעת. שלב קריטי לביסוס והרחבת monolayer חסיד הוא דיסוציאציה היעיל של neurospheres העיקרי. בעקבות מעבר, מספיק ניתק neurospheres עלול ליצור neurospheres צף משני. …

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia grants (#568969 and 1004152).

Materials

1000 μL filtered pipette tip Axygen TF1000
150 mm petri dish BD Biosciences 351058
15mL centrifuge tubes Greiner Bio One 188271
200 μL filtered pipette tip Axygen TF200
24 well culture plate Greiner Bio One 662160
35 mm tissue culture dish BD Biosciences 353001
40 µm cell strainer BD Biosciences 352340
6 well culture plate BD Biosciences 351146
B-27 Supplement (50X) serum free Life Technologies  17504044
Basic fibroblast growth factor (bFGF) Life Technologies 13256029
Brain derived neurotrophic factor (BDNF) Millipore GF029
Collagen solution Stem Cell Technologies 04902 Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740 
DMEM (4.5g/L, D-glucose) 500mL Life Technologies  11960044 
DPBS Life Technologies 14190250
Epidermal growth factor (EGF) BD Biosciences 354001
F-12 nutrient mixture (Ham) (1X) Liquid Life Technologies 31765035
Fetal bovine serum (FBS) Life Technologies 16141079
Gelatin from Porcine Skin Type A Sigma-Aldrich G1890
L-alanyl-L-glutamine dipeptide (GlutaMAX) Life Technologies 35050061
Heparin sodium salt from (porcine) Sigma-Aldrich H314950KU
Laminin (mouse) Life Technologies 23017015
NCFC serum free medium (NeuroCult) Stem Cell Technologies 5720 Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740 
Proliferation NS-A (NeuroCult) Stem Cell Technologies 05773 Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771  from Stem Cell Technologies.
NSC basal medium (Rat; NeuroCult) Stem Cell Technologies 5770 Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771 
Penicillin/Streptomycin (5000 U/mL) Life Technologies 15070063
Povidone-iodine Munipharma Betadine
Trypan blue (0.4%) Life Technologies 15250061
Trypsin EDTA Life Technologies 25200056
Class II biological safety cabinet ThermoFisher Scientific Safe 2020 1.2
Brain knife (disposable) Macroknife
Cell culture incubator ThermoFisher Scientific HERAcell 150i
Centrifuge Hettich Universal 320R
Dumont #5 Forceps Dumont
Easypet Electric pipette Eppindorf 
Hemocytometer Boeco Bright-Line Improved Neubauer
Manual pipettes Eppindorf research
Oscillating bone saw 
Scalpel blades (No.21) Paramount
Scissors  Delta
Water bath Grant JB Aqua 18 plus

References

  1. Kheirbek, M. A., Hen, R. Dorsal vs ventral hippocampal neurogenesis: implications for cognition and mood. Neuropharmacol. 36 (1), 373 (2011).
  2. Tanti, A., Rainer, Q., Minier, F., Surget, A., Belzung, C. Differential environmental regulation of neurogenesis along the septo-temporal axis of the hippocampus. Neuropharmacol. 63 (3), 374-384 (2012).
  3. Eriksson, P. S., et al. Neurogenesis in the adult human hippocampus. Nat Med. 4 (11), 1313-1317 (1998).
  4. Bull, N. D., Bartlett, P. F. The adult mouse hippocampal progenitor is neurogenic but not a stem cell. J Neurosci. 25 (47), 10815-10821 (2005).
  5. Kempermann, G., et al. Why and how physical activity promotes experience-induced brain plasticity. Front Neurosci. 4 (189), 1-9 (2010).
  6. Golmohammadi, M. G., et al. Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain. Stem Cells. 26 (4), 979-987 (2008).
  7. Lowe, A., et al. Neurogenesis and precursor cell differences in the dorsal and ventral adult canine hippocampus. Neurosc. Lett. 593, 107-113 (2015).
  8. Sasaki, M., et al. MRI identification of dorsal hippocampus homologue in human brain. Neuroreport. 15 (14), 2173-2176 (2004).
  9. Powell, E. W., Hines, G. . The Hippocampus. , 41-59 (1975).
  10. Jung, M. -. A., et al. Canine hippocampal formation composited into three-dimensional structure using MPRAGE. J Vet Med Sci. 72 (7), 853-860 (2010).
  11. Reynolds, B. A., Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 255 (5052), 1707-1710 (1992).
  12. Ray, J., Raymon, H. K., Gage, F. H. Generation and culturing of precursor cells and neuroblasts from embryonic and adult central nervous system. Methods Enzymol. 254, 20-37 (1995).
  13. Oliver-Dela Cruz, J., Ayuso-Sacido, A. Neural stem cells from mammalian brain: isolation protocols and maintenance conditions. INTECH Open Access Publisher. , (2012).
  14. Pacey, L., Stead, S., Gleave, J., Tomczyk, K., Doering, L. Neural stem cell culture: neurosphere generation, microscopical analysis and cryopreservation. Nat Protoc. , (2006).
  15. Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay. J Vis Exp. (45), e2393 (2010).
  16. Walker, T. L., Kempermann, G. One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice. J Vis Exp. (84), e51225 (2014).
  17. Lim, J. -. H., Koh, S., Olby, N. J., Piedrahita, J., Mariani, C. L. Isolation and characterization of neural progenitor cells from adult canine brains. Am J Vet Res. 73 (12), 1963-1968 (2012).
  18. Herranz, C., et al. Spontaneously Arising Canine Glioma as a Potential Model for Human Glioma. J Comp Pathol. 154 (2), 169-179 (2016).
  19. Walton, R. M., Parmentier, T., Wolfe, J. H. Postnatal neural precursor cell regions in the rostral subventricular zone, hippocampal subgranular zone and cerebellum of the dog (Canis lupus familiaris). Histochem Cell Biol. 139 (3), 415-429 (2013).
  20. Walton, R. M., Wolfe, J. H. Abnormalities in neural progenitor cells in a dog model of lysosomal storage disease. J Neuropathol Exp Neurol. 66 (8), 760-769 (2007).
  21. Milward, E. A., et al. Isolation and transplantation of multipotential populations of epidermal growth factor-responsive, neural progenitor cells from the canine brain. J Neurosci Res. 50 (5), 862-871 (1997).
  22. Ray, J., Peterson, D. A., Schinstine, M., Gage, F. H. Proliferation, differentiation, and long-term culture of primary hippocampal neurons. Proc. Natl. Acad. Sci. 90 (8), 3602-3606 (1993).
  23. Palmer, T., Ray, J., Gage, F. FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci. 6 (5), 474-486 (1995).
  24. Zhu, G., Mehler, M., Mabie, P., Kessler, J. Developmental changes in progenitor cell responsiveness to cytokines. J. Neurosci. Res. 56 (2), 131-145 (1999).
  25. Whittemore, S. R., Morassutti, D. J., Walters, W. M., Liu, R. -. H., Magnuson, D. S. Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations. Exp. Cell. Res. 252 (1), 75-95 (1999).
  26. Kennedy, P. Postmortem survival characteristics of rat glial cells in culture. J Neurol Neurosurg Psychiatry. 50 (6), 798-800 (1987).
  27. Viel, J. J., McManus, D. Q., Cady, C., Evans, M. S., Brewer, G. J. Temperature and time interval for culture of postmortem neurons from adult rat cortex. J. Neurosci. Res. 64 (4), 311-321 (2001).
  28. Huat, T. J., et al. IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells. BMC Neurosci. 15 (91), 1-13 (2014).
  29. Supeno, N. E., et al. IGF-1 acts as controlling switch for long-term proliferation and maintenance of EGF/FGF-responsive striatal neural stem cells. Int. J. Med. Sci. 10 (5), 522-531 (2013).
  30. Ahmed, S., Reynolds, B. A., Weiss, S. BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J Neurosci. 15 (8), 5765-5778 (1995).
  31. Wohl, C. A., Weiss, S. Retinoic acid enhances neuronal proliferation and astroglial differentiation in cultures of CNS stem cell-derived precursors. J Neurobiol. 37 (2), 281-290 (1998).
  32. Araujo, D. M., Cotman, C. W. Trophic effects of interleukin-4,-7 and-8 on hippocampal neuronal cultures: potential involvement of glial-derived factors. Brain Res. 600 (1), 49-55 (1993).
  33. Jensen, J. B., Parmar, M. Strengths and limitations of the neurosphere culture system. Mol Neurobiol. 34 (3), 153-161 (2006).
  34. Singec, I., et al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods. 3 (10), 801-806 (2006).
  35. Conti, L., et al. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol. 3 (9), 283 (2005).
  36. Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. Adherent neural stem (NS) cells from fetal and adult forebrain. Cereb Cortex. 16, 112-120 (2006).
  37. Ma, W., et al. Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells. BMC Dev Biol. 8 (90), 1-13 (2008).
  38. Goffredo, D., et al. Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells. Cell Death Differ. 15 (12), 1847-1856 (2008).

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Cite This Article
Duncan, T., Lowe, A., Dalton, M. A., Valenzuela, M. Isolation and Expansion of Adult Canine Hippocampal Neural Precursors. J. Vis. Exp. (117), e54953, doi:10.3791/54953 (2016).

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