A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Summary

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Abstract

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 °C.

Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Introduction

Xenografts are useful tools to investigate the behavior of human cells in vivo. These models have provided invaluable information for a wide range of scientific topics, such as the biology of human stem cells¹, the observation of cellular events in real time², and the investigation of tumoral angiogenesis and metastasis³. In addition, several aspects of cancer biology, including the tumorigenesis of patient-specific xenografts, have been studied^4,5. Each of these xenograft models has their advantages and disadvantages and, thus, each one is better suited for specific scientific questions. Chick embryos are a popular developmental biology model as they are an accessible amniote model that is amenable to surgical manipulation. Heterologous grafts have allowed researchers to create precise fate maps⁶ or explore whether a trait is cell-autonomous or instructed by the environment^7,8. A similar rationale allows the chick embryo to be used as a xenograft model to study the behavior of human cells.

The embryonic environment actively orchestrates tissue morphogenesis with migration and differentiation signals, as well as cell-cell interactions. Thus, compared to adult xenograft models, the embryo provides a more instructive milieu to assay the behavior of grafted cells, for example, by mimicking signals present in adult stem cell niches (e.g., BMPs, WNTs, NOTCH, and SHH⁹). In addition, the absence of an adaptive immune system during early development allows xenografts to be performed without the risk of an immune response or rejection of the donor tissue¹⁰. Previous studies have investigated xenografts of human cells into chicken embryos for this purpose. The neurogenic potential of human stem cells has been assayed after injection into the neural tube or blood vessels¹¹ in addition to the integration of embryonic stem cells¹² and induced pluripotent stem cells¹³ into the embryo. Human melanoma cells have also been studied using the chick's embryonic environment, which revealed links between their tumorigenesis and the behavior of neural crest cells¹⁴, as well as the reprogramming of the tumor cells with the information from the embryo¹⁵. This paper describes a protocol that is especially suited for studying the behavior of human primary and heterogeneous cell populations.

In the last decades, the stromal component of diverse tissues has been studied as an autologous source of progenitor/stem cells and for its proangiogenic and immunoregulatory properties, previously known as "mesenchymal stem cells"^16,17,18. The first of these cell populations to be characterized was the bone marrow stromal/stem cell population (BMSCs), which have osteo-, adipo- and, to a lesser extent, chondrogenic potential in vivo^19,20. Adipose-derived stromal cells (ADSCs) are a heterogeneous population obtained by enzymatic digestion of the lipoaspirate or dermolipectomy samples, followed by isolation of the stromal-vascular fraction (SVF) and finally expansion in culture²¹. In culture, these cells are phenotypically characterized by markers shared with other mesenchymal populations, such as CD90, CD73, CD105, and CD44, unique markers such as CD36, and the absence of hematopoietic (CD45) or endothelial (CD31) markers²². Additionally, ADSCs have osteo-, adipo-, and chondrogenic potential in vitro, and the number of stem/progenitor cells in this population can be defined by the fibroblastoid colony-forming unit (CFU-F) assay²². In vivo, cells with the ADSC phenotype have been reported to exist in stromal²³ and/or perivascular²⁴ compartments. It is becoming increasingly clear that, despite sharing markers after in vitro culture, the stromal compartment of different tissues reflects intrinsic characteristics of a given organ, and these cell populations have distinct properties depending on their source^17,25,26,27. Furthermore, as these cells are isolated based on their adhesion to a cell culture dish, they may be composed of cells from diverse germ layers²⁸. Thus, employing a xenograft method to study the differentiation potential and tropism of stromal cells in an unbiased way can provide valuable information about these cell populations to guide the development of future cell therapies.

The protocol described here (Figure 1) is a xenograft method that takes advantage of the low cost and ease of manipulation of chick embryos. It has been previously used to study the behavior of human ADSC²⁹, skin fibroblasts²⁹, menstrual blood-derived stromal cells³⁰, and glioblastoma cells³¹. This method will include the transplantation of cells as spheroids³², which can be prepared from any population of adherent cells (Figure 2). Surgical procedures and the preparation of custom surgical materials-the microscalpels and glass capillaries-will also be described (Figure 3). Human cells are detected in histological sections by hybridizing human-specific Alu probes (Figure 4), thus eliminating the need for prior labeling of the grafted cells. The representative results describe the behavior of human ADSC grafted both in the somitic region at the wing bud level (Figure 5, Figure 6, and Figure 7) and the first pharyngeal arch (Figure 8), as well as human primary glioblastoma spheroids grafted in the prosencephalon (Figure 8). Cell migration, differentiation, and interaction with chick embryonic tissues will be described, as well as suggested assays to further investigate cell behavior using co-staining or staining of adjacent sections.

Protocol

All in vivo procedures used in this study complied with all relevant experimental guidelines for animal testing and research, in accordance with the Brazilian experimental animal use guidelines (L11794). The protocols used for handling chicken embryos were all approved by the Ethics Committee on the Use of Animals in Scientific Experimentation (Health Sciences Centre of the Federal University of Rio de Janeiro). The use of human cells was approved by the Ethics Committee of the University Hospital Clementino Fraga Filho (numbers 043/09 and 088/04). Specific pathogen-free (SPF) eggs of White Leghorn chicken (Gallus gallus) were used.

1. Preparation of cell spheroids

NOTE: Cell spheroids can be prepared with a wide range of cell types as long as they are adherent. For this protocol, human adipose-derived stromal cells (ADSCs) were isolated as previously described^21,27 will be used (Figure 2A). Cells were obtained by digesting adipose tissue fragments or lipoaspirates with collagenase IA for 1 h at 37 °C under agitation, followed by plating at 1−2 × 10⁴ cells/cm² and overnight incubation. Non-adherent cells were discarded, and the adherent cells were expanded for 3-6 passages. ADSCs were homogeneous for the expression of the surface antigens CD105, CD90, CD13, and CD44 and negative for hematopoietic antigens CD45, CD14, CD34, CD3, and CD19²⁷. While the method described here can be performed easily with minimal materials beyond what is routinely used for cell culture, optimal aggregation time and the need for partial dissociation should be determined empirically. Alternative methods for preparing cell spheroids may be employed, such as the hanging-drop method³³ or agarose-coated wells³⁴; see the discussion for more details. All procedures should be performed on a clean bench employing aseptic techniques.

1. Warm up sterile PBS (phosphate-buffered saline), culture medium, and trypsin solution to 37 °C before cell manipulation.
2. Culture ADSCs in low glucose DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Use a confluent 25 cm² flask of ADSCs for the preparation of two plates of spheroids. Discard the culture medium and wash the culture dish three times with an appropriate amount of sterile PBS (5 mL PBS for each wash if a 25 cm² cell culture flask is used).
3. Add enough trypsin solution (containing 0.78 mM EDTA) to cover the cells (1 mL of trypsin solution if a 25 cm² cell culture flask is used) and let the culture flask sit for 5 min at room temperature.
4. Gently pipette the cells up and down to dissociate them. Add the same amount of culture medium containing FBS to inactivate the trypsin, and transfer the cell suspension to a 15 mL conical tube.
5. Centrifuge for 10 min at 180 × g. Discard the supernatant, and tap the tube to loosen the pellet.
6. Resuspend the cells in 500 µL-2 mL of culture medium adequate for the cell type (see step 1.2).
   NOTE: The minimum concentration is 5 × 10⁵ cells/mL for adherent cells such as ADSCs and fibroblasts. A confluent 25 cm² flask of ADSCs should be resuspended in 2 mL of medium and plated in 2 Petri dishes, using 1 mL each. Less adherent cells, such as glia, may benefit from plating at ~2 × 10⁶ cells/mL in a lower volume.
7. Carefully transfer the cell suspension to one side of a sterile 60 mm Petri dish (uncoated and untreated, such as those used for preparing agar plates). Try to restrict the culture medium to the smallest possible area (Figure 1B) by propping the dish over a piece of folded, clean gauze to keep it tilted in the incubator.
8. Incubate the cell suspension at 37 °C, 5% CO₂, until cell aggregates are formed (6-8 h after plating the ADSCs).
   NOTE: Spheroid aggregation time may vary according to cell type.
9. If required (see discussion), partially dissociate large cell aggregates by gently pipetting up and down using 1000 µL tips. Dissociate ADSC spheroids 6-8 h after plating.
10. Place the plate in the incubator until the spheroids are ready to be transplanted: round with defined edges and not easily dispersed (Figure 1C,D).
    ​NOTE: ADSC spheroids are ready 2 days after partial dissociation.

2. Transplantation of spheroids into chick embryos

1. Preparation
   1. Prepare chicken eggs by incubating 15-30 eggs in a humid incubator at 37.5 °C until the desired Hamburger-Hamilton (HH) stage is reached³⁵: 40-45 h for grafts in somites at the limb bud level (HH11-12 or 13-19 somites) or 29-33 h for grafts at the cephalic region (HH8-9 or 5-8 somites). Position the eggs horizontally, and draw a line with a pencil on the top of the eggs before incubation (Figure 3D).
      NOTE: Incubation times may vary slightly according to individual incubators. Drawing a line will ensure that the top portion of the egg, where the embryo is found, can be easily identified if the egg is rotated before the experiment.
   2. Prepare sharp needles ("microscalpels") by attaching a sewing needle to a metal needle holder (Figure 3B). Using an oiled whetstone, sharpen the needle until it resembles a small scalpel with a thin edge (Figure 3C). Alternatively, prepare a sharp tungsten needle by electrolysis³⁶.
   3. Prepare glass capillaries using the flame of a Bunsen burner to briefly melt the thin portion of a glass Pasteur pipette. Remove the molten portion from the flames and stretch it to form a thin capillary. Carefully divide the capillary into two parts by breaking it with microforceps. Prepare a thin capillary for India ink injection and a thick capillary for transferring spheroids (Figure 3B).
      NOTE: By varying the pulling strength, it is possible to create different capillary gauges. Capillaries should be prepared just before the surgery.
   4. Prepare the working surface and other materials for egg manipulation (Figure 3A). Sterilize all surgical materials in a 100 °C oven overnight or with 70% ethanol immediately before use. Warm sterile PBS to 37 °C. Attach a 200 µL tip (with barrier) to the aspirator tube assembly for India ink injection and transferring spheroids.
      NOTE: Use tips with barriers when transferring spheroids of human cells.
   5. Just before the experiment, add 2 drops of India ink stock solution to the 30 mm Petri dish and mix it with 1 mL of sterile PBS. Remove the spheroid plate from the incubator and place it over an icebox for the rest of the experiment.
2. Preparation of each egg
   1. Remove one egg from the incubator and place it over an egg holder. Make a small hole in the sharp end of the egg (Figure 2D) and aspirate 1.5 mL of albumen using a syringe and needle. Insert the needle perpendicular to the egg to avoid damaging the egg yolk. Seal the hole using adhesive tape. Optionally, repeat this step for all eggs before the experiment, and reincubate the remainder of the eggs.
   2. Carefully cut open a window on the top of the egg using scissors (Figure 3D). Using the pipette, add 1-2 drops of PBS over the yolk. If necessary, remove large albumen bubbles (such as the ones seen in Figure 3F) using the pipette.
   3. Attach the thin capillary to the aspirator tube assembly. Fill the glass capillary partially with the India ink solution by aspiration. Inject enough India ink into the yolk under the embryo until the embryonic structures are seen (Figure 3E,F).
   4. Using the stereomicroscope, count the number of somites (Figure 4A). Individually number the eggs using a pencil (Figure 4B).
3. Spheroid transplantation
   1. Identify the region of the graft: paraxial mesoderm at the wing bud level (presomitic mesoderm of the presumptive 15^th to 20^th somites³⁷) (Figure 3G and Figure 5A) or presumptive first pharyngeal arch region (between the ectoderm and endoderm lateral to the posterior mesencephalon/first rhombomere³⁸) (Figure 8A).
   2. Cut the vitelline membrane over the target region using a microscalpel.
   3. Using a pair of microscalpels, make a shallow cut in the region where the spheroid will be implanted (Figure 3H).
      NOTE: Avoid damaging the underlying endoderm; otherwise, the yolk will leak (if this happens, discard the egg).
   4. Remove the thin capillary from the aspirator and replace it with the thick capillary. Observe the spheroids under the stereomicroscope and choose a spheroid approximately the same size as the somite (Figure 2D). Aspirate this spheroid into the capillary.
   5. Gently deposit the spheroid next to the cut region (Figure 3H). Using the sharp needles, push the spheroid into the cut region (Figure 3I,J).
   6. Add 1-2 drops of PBS over the embryo using the pipette to ensure that the spheroid is firmly inserted.
      NOTE: If the graft is dislodged, a new spheroid should be transplanted (steps 2.3.4 and 2.3.5).
   7. Clean any albumin leak from the eggshell using tissue paper to ensure that the egg can be completely sealed, avoiding contamination. Seal the window in the egg using adhesive tape.
   8. Take note of the grafted region (Figure 4A). Carefully return the egg to the humid incubator at 37.5 °C to avoid dislodging the inserted spheroid.
   9. Incubate the egg until the desired stage.
      NOTE: When the xenograft is performed into somites at the limb bud level, migration and cell death have been successfully assayed in 3.5-day-old embryos (HH21), and cell differentiation and tropism have been observed in 6-day-old embryos (HH29) (Figure 5A). Integration into host tissues has been performed in 8-day-old embryos as well (HH33). Donor cells grafted to the pharyngeal arch territory have been studied in 4.5-day-old embryos (HH25) (Figure 8A).

3. Tissue dissection, fixation, processing, and preparation of histological sections

1. Dissection and fixation
   1. Prepare the fixative (at least 1 mL/embryo): ethanol 100%-formaldehyde 37%-glacial acetic acid in a 6:3:1 ratio. Clean the work surface, a pair of microforceps, surgical scissors or iris scissors, and a slotted spoon. Fill a 60 mm glass Petri dish with cold PBS and place it under a stereomicroscope.
   2. Open the egg by cutting out the adhesive tape.
   3. Cut the membranes around the embryo and remove it using the slotted spoon.
   4. Transfer the embryo to the Petri dish. If the embryo is 5-day-old (HH26) or older, promptly decapitate it before any manipulation ex ovo.
   5. Remove any remaining membranes using microforceps and scissors. Gently agitate the specimen in PBS to wash away any remaining yolk droplets.
   6. If the xenograft was performed at the wing level, discard the head. If cells were grafted to the cephalic region, cut and discard the lower half of the body, ensuring that the cardiac region is kept intact.
   7. Transfer each specimen to a separate 2.0 mL tube containing 1 mL of the fixative. Identify the tubes individually as before (Figure 4B). Incubate them overnight at 4 °C with agitation.
   8. Use a pellet of cells or spheroids (Figure 4F) as a positive control. For this, centrifuge a cell suspension (step 1.5) or cell spheroids (step 1.10) at 180 × g for 10 min in a 1.5 mL tube, discard the culture medium, and add 1 mL of the fixative without disturbing the pellet. Incubate the tube overnight at 4 °C without agitation, and proceed in the same manner as for the chick samples.
2. Dehydration and embedding
   1. Dehydrate the embryos by successive washes of 1 mL of 70%, 80%, 90%, and 100% ethanol in 1x PBS for at least 1 h each with agitation.
   2. Incubate the samples overnight in 1 mL of 100% ethanol at room temperature with agitation.
   3. Incubate the samples in 1 mL of 100% xylene for 1 h in a fume hood. Repeat two more times.
   4. Transfer the contents of the tube to the staining block and discard the xylene. Add enough molten paraffin to cover the embryo and cover the staining block with its glass lid. Incubate overnight in a 65 °C oven.
   5. Prepare a warm plate and a mold for paraffin blocks for embedding.
      NOTE: A pair of stretched-out paper clips are useful for positioning the embryo.
   6. Fill the mold with molten paraffin and transfer the embryo to it. Position the embryo to obtain a transverse section of the trunk (Figure 5A) or a coronal section of the head (Figure 8A).
   7. Insert a paper name tag in the paraffin, opposite the surface to be sectioned, to help cut the embryo in the correct orientation (Figure 4C).
   8. If being used, place the embedding cassette over the sample. Let the block cool down completely before removing it from the mold. Alternatively, attach the hardened paraffin block to the cassette or other block holder using molten paraffin and let it cool down again.
3. Sectioning
   1. Prepare the materials for sectioning. Warm up a hot plate to 42 °C and cover a cardboard or Styrofoam plate with clean aluminum foil approximately 30 cm x 20 cm. Clean all surfaces before use.
      NOTE: Use gloves to avoid contamination with nucleases, especially if some sections will be used for RNA in situ hybridization. Avoid using a histology bath to stretch the paraffin sections for the same reason unless the water and surfaces can be thoroughly cleaned beforehand.
   2. Trim the excess paraffin with a microtome blade. Create a bevel on both sides, in a trapezoidal shape, for easy separation of each section using a scalpel blade in a later step (Figure 3A).
   3. Attach the block to the microtome. Position the block carefully to ensure that the left and right sides are parallel to each other. Perform a finer adjustment after cutting a few sections of the sample and observing them under a microscope.
   4. When the target region has been reached (either the limb bud or first pharyngeal arch), cut 7 µm sections and place the paraffin ribbons over the plate covered with aluminum foil. Section the whole target region (Figure 4C).
   5. For preparing serial sections, cover the adequate number of slide glasses with drops of sterile deionized water. Transfer individual sections to the slides sequentially, using brushes and a scalpel. Transfer adjacent sections to different slides to create serial sections (Figure 4D). Prepare series of 3 slides for 3.5-day-old, 4 slides for 4.5-day-old, and 5 slides for 6-day-old embryos. Stain adjacent sections with different probes, antibodies, or classical histological stains.
      NOTE: The same series may contain multiple adjacent slides if a large portion of the embryo is sectioned. Multiple sections can be transferred to each slide, keeping some space between sections to allow them to stretch. Do not place the slide on the warm plate yet.
   6. After all sections have been transferred to the slides, add more water until all the sections are floating over a single water drop. Place the slide on a warm plate and let the sections stretch. Remove the slide from the warm plate, and remove the water by tilting the slide glass carefully against tissue paper.
      ​NOTE: Do not let the sections directly touch the glass slide before they stretch.
   7. Let the slides dry overnight in a 37 °C incubator.

4. Synthesis of digoxigenin-labeled Alu probes by polymerase chain reaction (PCR)

1. Prepare a stock solution of the following human-specific primers³⁹: AluFw: 5'-CGA GGC GGG TGG ATC ATG AGG T-3' and AluRev: 5'-TTT TTT GAG ACG GAG TCT CGC-3'.
2. Prepare 50 µL of the following PCR reaction⁴⁰: 1x PCR buffer, 2.0 mM MgCl₂, 0.1 mM dCTP, 0.1 mM dGTP, 0.1 mM dATP, 0.065 mM dTTP, 0.035 mM dig-11-dUTP, 0.4 µM AluFw primers, 0.4 µM AluRev primers, 0.05 U/µL Taq polymerase, and 1 ng/µL human genomic DNA.
3. Run the PCR using the following settings: initial denaturation at 94 °C for 4 min followed by 40 cycles of 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s, followed by a final denaturation of 72 °C for 5 min.
4. Measure the probe concentration using a spectrophotometer. Store the probes at -20 °C.
   ​NOTE: The PCR product should be 200-300 bp long after electrophoresis in a 2% agarose gel²⁹.

5. Section in situ hybridization with Alu probes

1. Day 1: Permeabilization and hybridization
   NOTE: A sterilized slide glass jar should be used for all steps performed on day 1.
   1. Preheat one wash of PBT (0.1% Tween 20 in PBS) in a 37 °C water bath and prepare a moist chamber with 50% formamide/50% deionized water.
      NOTE: Calculate the volume of all washes based on the glass jar size. Unless specified, all washes are performed by immersion of the slides in the solution.
   2. Remove the paraffin by three successive washes in xylene, 5 min each, in a fume hood.
   3. Rehydrate the series by two washes in 100% ethanol for 5 min each, followed by 90/70/30% ethanol washes in PBS for 2 min each.
   4. Wash in PBT (0.1% Tween 20 in PBS) three times for 5 min each.
   5. For permeabilization, add 2 µg/mL Proteinase K to the preheated PBT, immerse the slides in the solution, and incubate them for 14 min in a 37 °C water bath.
   6. Fix the sections by immersion in 4% paraformaldehyde/PBS for 20 min at room temperature.
   7. Wash with PBS for 5 min.
   8. After wiping excess PBS from each slide, cover the sections with 300 µL of hybridization buffer (50% deionized formamide, 4x SSC pH 5.0, 1x Denhardt's solution, 5% dextran sulfate, 100 µg/mL salmon sperm DNA) (Table 1). Incubate for 1 h at 42 °C in the formamide chamber.
   9. Prepare a solution of 0.2 ng/mL Alu probe in hybridization buffer. Tip the slide to remove the hybridization buffer and add 120 µL of the Alu probe solution over the sections. Cover with a glass coverslip, taking care to avoid bubbles.
      NOTE: Parafilm should not be used to cover the slides in this step, as it will melt at temperatures above 70 °C.
   10. Heat the slides on a hot plate at 95 °C for 5 min.
       NOTE: Do not breathe the formamide fumes.
   11. Incubate the slides at 42 °C in the formamide chamber overnight.
2. Day 2: Stringency washes and immunohistochemistry
   1. Prepare 20x SSC buffer (3 M NaCl, 0.3 M sodium citrate, pH 7.5) (Table 1). Preheat two washes of 0.1x saline sodium citrate (SSC) buffer, pH 7.5, to 42 °C.
   2. Fill a slide glass jar with 2x SSC buffer, pH 7.5. Gently place the slides in the solution and wait for the coverslips to detach themselves. Remove the coverslips using pincers and incubate the slides in 2x SSC buffer for 5 min at room temperature.
   3. Rewash the slides with 2x SSC buffer, pH 7.5, for 5 min at room temperature.
   4. Wash the slides two times with 0.1x SSC buffer, pH 7.5, at 42 °C for 5 min each.
   5. Wash the slides two times with MABT (maleic acid buffer with Tween; 0.1 M maleic acid, 0.15 M sodium chloride, 0.1% Tween 20, pH 7.5) (Table 1) for 30 min each at room temperature.
   6. Wipe excess buffer from each slide and cover the sections with 400 µL of blocking solution (10% inactivated normal goat serum, 2% blocking reagent in MABT). Incubate for 2 h in a humid chamber (prepared with deionized water) at room temperature.
   7. Tip the slides to remove the liquid and add 150 µL of Fab anti-DIG fragments conjugated to alkaline phosphatase at a 1:2,000 dilution in blocking solution. Cover with a glass coverslip or parafilm and incubate in the humid chamber at 4 °C for 16 h.
3. Day 3: Color development
   1. Gently remove the coverslips or parafilm inside a jar with MABT (as in step 5.2.2), and incubate the slides in MABT for 30 min at room temperature.
   2. Wash with MABT three more times for 30 min each.
   3. Wash with MAB (maleic acid buffer; 0.1 M maleic acid, 0.15 M sodium chloride, pH 7.5) (Table 1) for 30 min.
   4. Wash two times with NTM (NaCl-Tris-MgCl₂ buffer; 100 mM Tris-HCl pH 9.5, 100 mM sodium chloride, 50 mM magnesium chloride) (Table 1) for 10 min each.
   5. Add the staining solution (0.45 µL/mL 4-Nitro blue tetrazolium chloride, 3.5 µL/mL 5-bromo-4-chloro-3-indolyl phosphate p-toluidine in NTM) (Table 1) to the staining jar and immerse the slides in the solution. Cover the jar with aluminum foil and let the color develop overnight at 37 °C.
4. Day 4: Counterstaining and mounting
   1. Wash the slides three times with PBS at room temperature for 10 min each.
      NOTE: Ensure that the staining solution is disposed of properly as halogen waste.
   2. Mount the slides as they are by adding drops of an aqueous mounting media and covering the sections with a glass coverslip. Alternatively, proceed to either nuclear fast red or Alcian blue counterstaining.
   3. Wipe away excess PBS and add 500 µL of a nuclear counterstaining solution of 0.1% nuclear fast red⁴¹ (Table 1) over the sections and incubate the slides in a humid chamber for 10 min. Tip to remove excess nuclear fast red and proceed to dehydration (step 5.4.5).
   4. Immerse the sections in 0.5% Alcian blue solution (cartilage counterstain)⁴² for 10 min; rinse in distilled water and incubate in 1% phosphomolybdic acid for 10 min. Rinse with water again and proceed to dehydration.
      NOTE: If the phosphomolybdic acid wash is omitted, Alcian blue becomes a nuclear counterstain.
   5. Dehydrate in 70/95/100/100% ethanol for 5 min each.
   6. Wash with xylene three times for 5 min each time in a fume hood.
   7. Mount with a xylene-based mounting medium and glass coverslips.
   8. Let the slides dry for at least 16 h in a fume hood.

6. Image acquisition

1. After the mounted slides are thoroughly dried, examine all sections for the presence of grafted human cells using an upright brightfield microscope (Figure 4E). Look for blue-purple Alu-positive cells (Figure 4F,G) and mark the sections containing Alu-positive cells using a marker pen (Figure 4E).
   NOTE: Marking the sections with Alu-positive cells will make it easier to find them while photographing them and comparing adjacent slides stained with other markers.
2. Take care to photograph sections containing the xenograft in the same order as their placement on the slide (antero-posterior direction). Name each photo with all relevant information, such as [date of the xenograft]_[cell type]_[embryonic age]_[probe type]_[section number]_001. Include metadata files to add the scale bar later.
3. If other slides from the same series are stained with another marker, e.g., RNA in situ hybridization or immunohistochemistry, photograph and name adjacent sections (Figure 6) in the same manner.

Results

Identification of Alu-positive ADSCs in histological sections
Alu sequences are repetitive elements that comprise ~10% of the human genome and thus are excellent targets for identifying human cells in a species-specific manner⁴³. In situ hybridization with DNA probes can be used to identify genomic elements on histological sections, including primary human cells^29,

Discussion

The protocol described here (Figure 1) presents a feasible option for screening the behavior of primary populations of human cells in vivo, using chick embryos as a model. This paper describes the formation of cell spheroids (Figure 2), transplantation of the spheroid into the chick embryo (Figure 3), processing of specimens and in situ hybridization (Figure 4), representative results ...

Materials

Name	Company	Catalog Number	Comments
Animals
Gallus gallus eggs	Granja Tolomei	SPF-free	White leghorn chicken
Reagents
Alcian Blue 8GX	Sigma-aldrich	A5268
AluFw primers	Sigma-aldrich	OLIGO	5’-CGA GGC GGG TGG ATC ATG AGG T-3’
AluRev primers	Sigma-aldrich	OLIGO	5’-TTT TTT GAG ACG GAG TCT CGC-3’
Aluminum sulphate	Sigma-aldrich	368458	For Nuclear fast red solution preparation
Anti-Digoxigenin-AP, Fab fragments	Roche	11093274910	Antibody Registry ID: AB_514497
Anti-Human Natural Killer 1 antibody (HNK1, CD57)	Developmental Studies Hybridoma Bank	3H5	Antibody Registry ID: AB_2314644
Anti-mouse, goat IgM-HRP	Santa Cruz Biotechnology	sc-2973	Antibody Registry ID: AB_650513
Anti-mouse, goat IgG (H+L)-HRP	Novex	G-21040	Antibody Registry ID: AB_2536527
Anti-Smooth Muscle Actin/ACTA2 antibody	Dako	M085129	Antibody Registry ID: AB_2811108
Aquatex	Merck	1085620050	Aqueous mounting agent
5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP)	Sigma-aldrich	B8503-100MG
Blocking Reagent	Roche	11096176001
Citric acid	VETEC	238	For SSC buffer preparation
Collagenase type IA	Sigma-aldrich	SCR103
dCTP, dGTP, dATP, dTTP set	Roche	11969064001
Denhardt solution 50X	Invitrogen	750018	For hybridization buffer preparation
Dextran sulphate sodium salt	Thermo Scientific	15885118	For hybridization buffer preparation
DIG RNA Labeling Mix	Roche	11277073910	Contains Dig-11-dUTP
DMEM low-glucose	Sigma-aldrich	D5523
3,3′-Diaminobenzidine tetrahydrochloride (DAB)	Sigma-aldrich	D5905-50TAB
N,N-Dimethylformamide (DMF)	Sigma-aldrich	227056	For NBT and BCIP solution preparation
Ethylenediaminetetraacetic acid (EDTA)	Sigma-aldrich	E6758	For trypsin solution preparation
Entellan new	Merck	107961	Non-aqueous mounting medium
Ethanol	Proquímios	N/A
Fetal bovine serum	ThermoFisher	12657029	Inactivate at 56 °C before use
Formaldehyde 37% solution	Proquímios	N/A
Formamide	Vetec	V900064
Glacial acetic acid	Proquímios	N/A
India ink	Pelikan	221143
L-glutamine solution (200 mM)	Gibco	25030-149
Magnesium chloride	Merck	8147330100	For NTM buffer preparation
Maleic acid	Sigma-aldrich	M0375-500G	For MAB buffer preparation
Methanol	Proquímios
Normal Goat Serum	Sigma-aldrich	NS02L	Inactivate at 56 °C before use
4-Nitro blue tetrazolium chloride (NBT)	Roche	11585029001
Nuclear fast red	Sigma-aldrich	60700
Paraplast Plus	Sigma-aldrich	P3558
Penicillin G sodium salt	Sigma-aldrich	P3032
Phosphate buffered saline (PBS)	Sigma-aldrich	P3813
Phosphomolybdic acid	Merck	100532
Proteinase K	Gibco BRL	25530-015
Salmon sperm DNA	Invitrogen	15632011	For hybridization buffer preparation
Sodium chloride	Sigma-aldrich	S9888	For SSC, MAB and NTM buffer preparation
Streptomycin Sulfate	Sigma-aldrich	S6501
Taq Polymerase kit	Cenbiot Enzimas	N/A
Tris-HCl	Sigma-aldrich	T5941
Trypsin	Sigma-aldrich	T4799
Tween 20	Sigma-aldrich	P1379
Xylene	Proquímios	N/A
Microscope and equipments
Axioplan upright microscope	Carl Zeiss Microscopy	N/A
Axiovision software	Carl Zeiss Microscopy	N/A
Cell incubator	ThermoForma	3110
Egg incubator- 50 eggs	GP
Gooseneck lamp	Biocam	N/A	For egg manipulation
Fiji software; Cell Counter plugin	ImageJ	https://imagej.net/software/fiji/
Laminar flow hood	TROX	1385
Nanodrop Lite	Thermo Scientific	ND-LITE-PR
Rotary microtome	Leica Biosystems	RM2125 RTS	For sectioning
Stereomicroscope	Labomed	Luxeo 4D	For egg manipulation
Sterilization oven	REALIS	7261690	For sterelization of surgical materials
Consumables
0.2 mL (PCR) polypropylene centrifuge tubes	Eppendorf	30124707
15 mL polypropylene conical centrifuge tubes	Corning	CLS430791
1.5 mL polypropylene centrifuge tubes	Axygen	MCT-150-C
2 mL polypropylene centrifuge tubes	Axygen	MCT-200-C
50 mL polypropylene conical centrifuge tubes	Corning	CLS430829
Barrier (Filter) Tips, 200 μL size	Invitrogen	AM12655	For egg manipulation
Excavated Glass Block (Staining Block) with Cover Glass	Hecht Karl	42020010
Embedding cassettes	Simport	M480	Used as a paraffin block holder
Glass coverslides, 24 x 40 mm	Kasvi	K5-2440
Glass Pasteur pipettes 230 mm	NORMAX	5426023	For preparation of glass capillaries
Microtome blades	Leica Biosystems	HIGH-PROFILE-DISPOSABLE-BLADES-818	For sectioning
Parafilm M	Parafilm	P7793
Plastic Petri dish, 30 mm	Kasvi	K13-0035	For egg manipulation
Plastic Petri dish, 60 mm	Prolab	0303-8	For cell spheroids preparation. Should not be treated for cell adhesion.
Silanized glass slides (Starfrost)	Knittel Glass	198	For sectioning
Syringe 1 mL , Needles 26 G (0.45 x 13 mm)	Descarpack	32972	For egg manipulation (albumen aspiration)
Surgical tools
Aspirator tube	Drummond	2-000-000	For egg manipulation
Dissection scissors	Fine Science Tools	14061-11	For egg manipulation
Microforceps (tweezers)	Fine Science Tools	00108-11	For egg manipulation and preparation of glass capillaries
Needle holder (adjustable dissection needle chuck)	Fisherbrand	8955	For egg manipulation
Oil whetstone, 10.000 grit	N/A	N/A	For sharpening needles
Pair of small paint brushes	N/A	N/A	For handling paraffin sections. Any brand may be used.
Sewing needles	N/A	N/A	For sharpening into microscalpels. Any brand may be used.
Sterile disposable scalpel No. 23	Swann-Norton	110	For sectioning
Surgical scalpel handle	Swann-Norton	914	For sectioning
Wecker iris scissors, sharp/sharp	Surtex	SS-641-11	For egg manipulation