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, e....

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.

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

List of materials used in this article

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