Improved Method for Ex Ovo-Cultivation of Developing Chicken Embryos for Human Stem Cell Xenografts

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.


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 et al., motor neurons derived from embryonic stem cells as well as induced motor neurons reprogrammed from mouse and human fibroblasts were shown to integrate after transplantation into the chicken neural tube [4,5].
For the investigation of developing chicken embryos, Auerbach and coworkers designed a method allowing longterm cultivation of chicken embryos in an ex ovo-setup [6]. In 1989, the containment for ex ovo-cultivation was improved concerning the short-term survival using a plastic cup covering the developing embryo with a petri dish [7]. This method permits easy access to the embryo as well as to the blood vessels of the chorioallantoic membrane (CAM). Besides the observation of the development, ex ovo-cultivated chicken 2 Stem Cells International Figure 1: Schematic view of initial steps in ex ovo-cultivation of chicken embryos. Prior to transfer, chicken embryos were incubated at 37.8 ∘ C for 48 hours. Eggs were gently opened using a jigsaw. The indentation was expanded, and the contents were carefully transferred onto the support film. Ground eggshell of several eggs was added to the albumin and the experimental setup was covered using a bacterial dish. embryos can be used for the investigation of toxicity of different substances in a vertebrate model. In this context, a shellless cultivation method was used to observe the influence of nicotine and cigarette smoke in developing chicken embryos [8]. In addition, the effects of acute glucose toxicity could be assessed in shell-less chicken embryo cultures [9].
Recently, Yalcin and colleagues described an ex ovocultivation method of chicken embryos, which is suitable for microsurgical and imaging applications [10]. However, eggs were incubated for 72 hours previous to the transfer into the ex ovo-setup, and if cultivated beyond embryonic day (E) 7, crushed eggshell was added to achieve HH38, correlating with E12.
Here, we describe an inexpensive reusable shell-less cultivation method for chicken embryos in a broadly available containment. Using a defined amount of water and ground eggshell, we demonstrate for the first time the survival of ex ovo-cultivated embryos for at least 13 days up to E15 and HH stage 41. Moreover, in contrast to methods previously described, the herein presented method supports xenografts into late stages of ex ovo-cultivated chicken embryos.

Ex Ovo-Cultivated Chicken Embryos Reveal Normal Morphological Development for 13 Days In
Vitro. For the herein described ex ovo-cultivation method, egg contents of chicken eggs preincubated for 48 hours were gently transferred into a readily prepared shell-less containment, as demonstrated schematically in Figure 1. Development and morphology were compared to "normal stages of the chicken embryo" described by Hamburger and Hamilton [1]. As shown in Figure 2, embryos were easily detectable at E4 revealing normal development. During incubation, the yolk expanded on the support film of the ex ovo-containment, and the blood vessels started to span smoothly over the yolk. Furthermore, continuous observation showed normal morphological development of embryos up to E15, correlating with HH stage 41.

Ex Ovo-Cultivated Chicken Embryos Show Improved Survival without Significant Differences in Size and Weight.
To investigate the size and weight, in ovo-and ex ovocultivated developing chicken embryos were sacrificed and compared at E10 as well as at E13 and E15 ( Figure 3). Here, no significant differences in size were detectable between shell-less and traditionally cultivated chicken embryos. In addition, the embryos showed no significant differences in weight at E13 and E15 when incubated in a shell-less containment. We estimated the survival rate for embryos cultivated for 13 days in vitro (4 cohorts, 11 embryos each). Starting with easily visible chicken embryos, the survival rate was measured starting at E4 of ex ovo-cultivation. The addition of cell culture medium did not enhance the survival rate of chicken embryos (data not shown), which is contrary to the observations made by Auerbach and coworkers [6]. Importantly, cultivated in a humidified incubator at 37.8 ∘ C, more than 18% of the embryos were able to survive until E15.

Chondrogenesis, Osteogenesis, and Myelination of Nerves
Were Not Impaired by Ex Ovo-Cultivation. Development of in ovo-and ex ovo-cultivated chicken embryos was compared at given time points regarding chondrogenesis, osteogenesis, and myelination of the optic nerves. At E5, the vertebrae of chicken embryos started to undergo chondrification [11]. In contrast to E5 embryos, which did not show specific staining for cartilage, E10 chicken embryos were positive for Alcian blue staining suggesting that cartilage and bone tissue of the chicken embryo started to chondrify at this point of time (Figure 4). At E13 and E15 of development, no differences in chondrification between in ovo-and ex ovo-cultivated chicken embryos were observed. Regarding osteogenesis, chicken embryos at E5 showed no specific staining as expected (Figure 4). At E10, the wings, skull, and ribs began to ossify. However, in the respective tissues no specific staining for bone was observed in any of the analyzed chicken embryos. In contrast, at E13, ex ovo-and in ovo-cultivated chicken embryos showed distinct staining for bone at comparable amounts. Up to E15, ex ovo-cultivated embryos showed normal osteogenesis in comparison to in ovo-cultivated chicken embryos.
We applied Sudan Black B to specifically stain lipid-rich myelinated nerves within ex ovo-cultivated chicken embryos [12][13][14]. Focusing on the myelination of the optic nerves the orbital cavity was investigated at E10, E13, and E15. Chicken embryos at E10 did not show specific staining for myelinated optic nerves. However, in E13, chicken embryos staining of the optic nerve could be observed (Figure 4). A more distinct staining of the optic nerve at E15 indicated advanced myelination.

Xenografted ITSCs Integrate in the Basal Layer of the Epidermis of Ex Ovo-Cultivated Chicken Embryos.
For xenografts into developing chicken embryos, ITSCs were virally transduced using lentivirus harboring the lacZ-gene E4/div 2 E5/div 3 E6/div 4 E7/div 5 E8/div 6 E9/div 7 E10/div 8 E11/div 9 E12/div 10 E13/div 11 E14/div 12 E15/div 13 Development of ex ovo-cultivated chicken embryos over time leading to a deep blue color of the cell nuclei aftergalactosidase staining. Using the here-described ex ovocultivation method, labeled ITSCs were injected into developing chicken embryos as late as HH stages 26 to 28, correlating with E5 to E6. An adequate time span of up to 4 days allowed proper integration and differentiation of xenografted adult human stem cells. Subsequently, manipulated chicken embryos were sacrificed followed by fixation and staining for lacZ-positive ITSCs. Tissue containing lacZ-positive stem cells was sectioned and stained using specific antibodies. LacZ-positive ITSCs injected into lesioned developing chicken embryos remained positive for the neural crest stem cell-marker nestin after 4 days, as demonstrated in Figure 5. Furthermore, xenografted ITSCs showed expression of the ectodermal marker -III-tubulin suggesting partial phenotypic switch towards ectodermal lineage in vivo. Interestingly, transplanted ITSCs also showed expression of the basal cell-marker cytokeratin 14 (CK14) pointing towards a basal cell-like differentiation. This observation was underlined by bright field microscopy showing integration of ITSCs into the basal cell layer of the epidermis.
Taken together, virally transduced ITSCs expressing the lacZ-gene were able to integrate into late stages of the developing chicken embryos after xenografting in ex ovocultivated chicken embryos.

Discussion
The herein described ex ovo-cultivation system allows survival of chicken embryos for up to embryonic day 15 and microsurgical transplantation of human NCSCs into the developing embryo at late stages (E5-7). Cultivated chicken embryos showed normal development, as demonstrated by proper osteogenesis, chondrogenesis, and myelination of nerves, as well as no significant differences to the in ovoapproach regarding their size and weight.
Its cost efficiency make the chicken embryo ideal for investigation and manipulation of development processes using variety of experimental methods. However, most of the cultivation methods deal only with early stages of development since in ovo-experiments of late developmental stages are restricted by the necessity of windowing the shell as well as by strong vascularization and presence of membranes [15]. To investigate late developmental stages of chicken embryos, the shell-less or ex ovo-cultivation was established and subsequently improved regarding the short-term survival of chicken embryos [6,7]. In 1999, Brooks and coworkers studied angiogenesis in 10-days-old chicken embryos by using shell-less culture systems [16]. Moreover, shell-less chicken embryo cultures were used to investigate functional importance of N-cadherin in the developing chicken limb by application of monoclonal N-cadherin-specific antibodies [17].
Nevertheless, these studies described ex ovo-cultivation at early stages of developing chicken embryos, particularly, at age prior to or at day 3 of incubation. Hamamichi and Nishigori as well as Datar and Bhonde used late stages of ex ovo-cultivated chicken embryonic development to examine the influence of nicotine in E7 embryos and effects of acute glucose toxicity, respectively [8,9]. Recently, Leong and coworkers applied a chorioallantoic membrane (CAM) assay to investigate cell migration and metastatic growth of cancer cells in ex ovo-cultivated chicken embryo systems [18]. However, none of the studies applied microsurgical applications to the chicken embryo itself. Although Dhole and colleagues already reported an injection method into the vitreous of the eye of late stage ex ovo-cultivated chicken, the authors did not investigate behavior and survival of injected cells [19]. Apart from this, the survival rate of chicken embryos was postulated to be over 50% after transfer, but no time-dependent statistics were made. More recently, Yalcin and colleagues presented an ex ovo-culture system allowing the cultivation of chicken embryos up to HH stage 38, correlating with E12 [10]. Despite using microsurgical methods, those were only applied to early stages of developing chicken embryos without statistically analyzing the survival.
Extending these promising findings, the herein described ex ovo-cultivation method allows cultivation of chicken embryos starting at 48 hours (E2) of incubation up to E15 correlating with HH stage 41 and beyond. Interestingly, the survival rate of chicken embryos decreases over time We further describe for the first time the utilization of microsurgical applications for experiments on late stages of ex ovo-cultivated chicken embryos at E5 and later. In this context, lacZ-positive ITSCs were stained for the ITSCmarker nestin after transplantation and integration into developing chicken embryos. This observation is consistent with expression in their endogenous niche of the human inferior turbinate [3,20].
Although early stages of developing chicken embryos are used for the characterization of stem cells such as in case of chain migration of NCSCs, differentiated tissues of chicken embryos cultivated for a prolonged time more closely resemble late stages of development of an organism as in case of differentiated tissue of limbs. Therefore, information about the differentiation potential in already developed tissue such as bones, cartilage, the nervous system, or skin can be obtained.
Taken together, we describe an ex ovo-cultivation method suitable for long-term cultivation and investigation of chicken embryos. In a developmental context, normal growth was shown with regard to weight and size as well as cartilage, bone, and myelinated nerves of embryos. Moreover, the injection of human neural crest-derived ITSCs was performed to investigate the usability of the herein-described ex ovomethod with respect to microsurgical applications. Here, our