The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26–HH28/E5—E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages. 1. Introduction The chicken is a well-studied and cost-efficient model organism profiting from a great potential of in vivo manipulation techniques. As early as the 5th century B.C. Hippocrates and later on in the 4th century B.C. Aristotle studied embryonic development using chicken embryos. More than 2000 years later, in 1951, Hamburger and Hamilton classified the developmental stages of the chicken embryo in 46 HH stages [1] allowing temporally defined manipulations in developing embryos. Using this kind of age-classification several in ovo experiments such as investigations on neural crest cells (NCCs) and their migratory behavior in the avian embryos were performed [2]. In this regard, stem cells obtained from different animals or even of human origin can be characterized for their potential neural crest ancestry. In a recent study, we transplanted human inferior turbinate stem cells (ITSCs) into early chicken embryos (HH15–HH18) [3]. The injected ITSCs migrated laterally forming chains, a characteristic hallmark of neural crest cells. In other studies by Soundararajan et al. and Son
References
[1]
V. Hamburger and H. L. Hamilton, “A series of normal stages in the development of the chick embryo. 1951,” Developmental Dynamics, vol. 195, no. 4, pp. 231–272, 1992.
[2]
S. Krispin, E. Nitzan, Y. Kassem, and C. Kalcheim, “Evidence for a dynamic spatiotemporal fate map and early fate restrictions of premigratory avian neural crest,” Development, vol. 137, no. 4, pp. 585–595, 2010.
[3]
S. Hauser, D. Widera, F. Qunneis et al., et al., “Isolation of novel multipotent neural crest-derived stem cells from adult human inferior turbinate,” Stem Cells and Development, vol. 21, pp. 742–756, 2012.
[4]
P. Soundararajan, G. B. Miles, L. L. Rubin, R. M. Brownstone, and V. F. Rafuse, “Motoneurons derived from embryonic stem cells express transcription factors and develop phenotypes characteristic of medial motor column neurons,” Journal of Neuroscience, vol. 26, no. 12, pp. 3256–3268, 2006.
[5]
E. Y. Son, J. K. Ichida, B. J. Wainger et al., “Conversion of mouse and human fibroblasts into functional spinal motor neurons,” Cell Stem Cell, vol. 9, no. 3, pp. 205–218, 2011.
[6]
R. Auerbach, L. Kubai, D. Knighton, and J. Folkman, “A simple procedure for the long term cultivation of chicken embryos,” Developmental Biology, vol. 41, no. 2, pp. 391–394, 1974.
[7]
A. M. Jakobson, R. Hahnenberger, and A. Magnusson, “A simple method for shell-less cultivation of chick embryos,” Pharmacology and Toxicology, vol. 64, no. 2, pp. 193–195, 1989.
[8]
S. Hamamichi and H. Nishigori, “Establishment of a chick embryo shell-less culture system and its use to observe change in behavior caused by nicotine and substances from cigarette smoke,” Toxicology Letters, vol. 119, no. 2, pp. 95–102, 2001.
[9]
S. Datar and R. R. Bhonde, “Shell-less chick embryo culture as an alternative in vitro model to investigate glucose-induced malformations in mammalian embryos,” The Review of Diabetic Studies, vol. 2, pp. 221–227, 2005.
[10]
H. C. Yalcin, A. Shekhar, A. A. Rane, and J. T. Butcher, “An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications,” Journal of Visualized Experiments, vol. 44, article e2154, 2010.
[11]
R. Bellairs and M. Osmond, The Atlas of Chick Development, Elsevier, Boston, Mass, USA, 2005.
[12]
G. T. Filipski and M. V. H. Wilson, “Sudan black B as a nerve stain for whole cleared fishes,” Copeia, vol. 1984, pp. 204–208, 1984.
[13]
K. C. Nishikawa, “Staining amphibian peripheral-nerves with sudan black B: progressive versus regressive methods,” Copeia, vol. 1987, pp. 489–491, 1987.
[14]
J. J. Meyers, A. Herrel, and K. C. Nishikawa, “Comparative study of the innervation patterns of the hyobranchial musculature in three iguanian lizards: sceloporus undulatus, Pseudotrapelus sinaitus, and Chamaeleo jacksonii,” Anatomical Record, vol. 267, no. 2, pp. 177–189, 2002.
[15]
M. J. Korn and K. S. Cramer, “Windowing chicken eggs for developmental studies,” Journal of Visualized Experiments, no. 5, article e306, 2007.
[16]
P. C. Brooks, A. M. Montgomery, and D. A. Cheresh, “Use of the 10-day-old chick embryo model for studying angiogenesis,” Methods in Molecular Biology, vol. 129, pp. 257–269, 1999.
[17]
S. A. Oberlender and R. S. Tuan, “Application of functional blocking antibodies. N-cadherin and chick embryonic limb development,” Methods in Molecular Biology, vol. 137, pp. 37–42, 2000.
[18]
H. S. Leong, A. F. Chambers, and J. D. Lewis, “Assessing cancer cell migration and metastatic growth in vivo in the chick embryo using fluorescence intravital imaging,” Methods in Molecular Biology, vol. 872, pp. 1–14, 2012.
[19]
D. S. Dohle, S. D. Pasa, S. Gustmann et al., “Chick ex ovo culture and ex ovo CAM assay: how it really works,” Journal of Visualized Experiments, article e1620, 2009.
[20]
A. Minovi, M. Witt, A. Prescher et al., “Expression and distribution of the intermediate filament protein nestin and other stem cell related molecules in the human olfactory epithelium,” Histology and Histopathology, vol. 25, no. 2, pp. 177–187, 2010.
