Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Summary

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Abstract

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions^1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny^3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function^6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.

Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific "niche" cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)⁹ and RBPJ knock-out mice (lacking canonical Notch signaling)¹⁰ are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Introduction

Intravital multiphoton fluorescence microscopy (IVFM) is a powerful imaging technique that allows for the high-resolution, real-time imaging of tissues with depth up to 1mm, depending on the tissue. When applied to the mouse calvarium, it permits observing the behavior of the hematopoietic cells within the BM in a non-invasive manner up to 60-100 μm¹¹. This approach is used here to determine the kinetics of engraftment of normal myeloid progenitors in the BM of RBPJ knock-out mice lacking canonical Notch signaling.

Recent work from our group demonstrated that defective canonical Notch signaling in the BM microen....

Protocol

All procedures involving the use of animals were performed with authorization of the Animal Care and Use Committee of Indiana University School of Medicine. Ensure to adhere to the legislation on animal experimentation of the country where the work is performed.

1. Preparation of Mx1CreRBPJ^-/- Recipient Mice

1. Cross Mx1-Cre⁺ mice with RBPJ^lox/lox mice¹⁰ to obtain Mx1-Cre positive ^RBPJlox/lox mice¹² and Mx1-Cre negative RBPJ^lox/lox littermates to use as controls. Verify the genotype by PCR^10....

Results

Cohorts of 2 RBPJKO and 2 RBPJWT recipients were imaged in an individual imaging session at different time points: 24 h and 2, 4 and 6 weeks after transplantation of BM Lys-GFP cells (workflow is illustrated in Figure 1A).

In each mouse, images were acquired from 6 standard regions of the BM calvarium, identified by their position in relation to the bifurcation of the central vein (Figure 2A,

Discussion

This protocol describes an experimental design optimized to study the kinetics of hematopoietic cells engraftment by Intravital Florescent Microscopy. In this study, the expansion of myeloid progenitor cells in a WT BM or in a Notch signaling defective BM was tracked in the bone calvarium by following Lys-GFP positive myeloid cells after BMT into RBPJWT or RBPJKO recipients. This approach is proposed as a model that can be applied to address similar questions, for example: i) to determine the expansion and localization i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine cocktail	IU School of Medicine		Ketamine 90-100 mg/kg, Xylazine 2.5-5.0 mg/kg, Acepromazine 1.0-2.5 mg/kg
TRITC dextran	Tdb Consultancy	TD150-100mg	Other color dextran may be used.
Andis hair trimmer	Braintree Scientific	CLP-323 75
Gauze sponge	Med Vet International	PK224	4-ply, 2 x 2
Nair depilatory cream	Commercial store
Saline	Med Vet International	RXSAL-POD1LT	0.9% Sodium Chloride poly bottle
Insulin syringe	Fisher Scientific	14-826-79	28 g, 1/2 cc
Fine Forceps	Fine Science Tools	00108-11, 00109-11	straight forcep, angled forcep
Scissor	Fine Science Tools	15018-10
Needle holder	Fine Science Tools	12002-14
5-0 silk suture	Fisher Scientific	MV-682	Other non-absorbable suture may be used
WillCo- glass bottom dish	WillCo	GWSt-5040
Optical microscope oil	Leica
Stereotaxic stage insert	IU School of Medicine	Custom design
Olympus FV1000 confocal microscope system	Olympus
Olympus XLUMPLFL 20XW, NA 0.95 objective	Olympus
Small heating pad	Commercial store		Zoo Med reptile heating pad
Imaris 8.1 imaging software	Bitplane		3/4 D Image Visualization and Analysis software