This protocol was tested and optimized in the laboratory of Dr. David Scadden. Funding is provided by the Dana Farber Cancer Institute/Massachusetts General Hospital Clinical Hematology and Oncology Fellowship Program.

<ol>
<li> We begin the homing experiment by extracting cells to be used in the experiment. For this current experiment we are interested in finding out if the homing of whole bone marrow to the niches in the long bones of recipient animals is different for bone marrow cells extracted from WT animals such as C57BL/6J (WT) and those that are transgenic knockouts for Lysophosphatidic Receptor 1 (LPAR1) (KO).</li>
<li>Before we start the experiment, we will collect the materials we need for this experiment. In our lab all mouse dissections are carried out in a laminar flow tissue culture hood especially if the cells are going to be transplanted into a recipient animal instead of being used for ex-vivo analysis. We start by placing a blue sheet inside the hood. Other objects needed include a pair of forceps and a sharp pair of scissors which have been autoclaved , a sterile disposable scalpel and alcohol swabs. We will need modified PBS and a sterile 6 well plate, a mortar, pestle, 50cc blue topped conical tube and a 70uM disposable filter.</li>
<li>We start by taking one WT and one KO mouse.</li>
<li>These mice are euthanized by CO2 inhalation and then dipped in 70% isopropranolol for 5 seconds. This step prevents contamination of the cells of interest with mouse fur and in our hands has not led to any compromise in yield of cells or experimental results.</li>
<li>The mice are then dissected in turn using a pair of forceps and sharp scissors. We usually start with the WT mouse as a matter of routine. A small snip is made in the ventral skin of the mouse overlying the abdomen and the skin is then stretched using the hands. The skin comes off the hind limbs easily. The femur and tibia are then extracted taking care not to dislodge the head of the femur as this contains a large amount of bone marrow. The upper limbs are detached from the animal&rsquo;s torso by simply applying traction to them while keeping the torso immobile. This removes the upper limbs as one piece. The humeri are then extracted from the upper limbs.</li>
<li>The removed bones are placed in modified PBS which has been added to 6 well plates. Modified PBS is made by adding 2 ml of heat inactivated Fetal Bovine Serum and 2 cc of EDTA 0.5 M pH8. This solution is then filtered through a Nalgene 0.45 uM filter and can be stored at 4 degrees Celcius for at least a month.</li>
<li>It is essential to label the wells in the plate in order to be able to identify WT and KO bones correctly.</li>
<li>The mouse carcasses are removed and disposed off.</li>
<li>A new sheet is placed in the hood and the bones are cleaned using a disposable scalpel.</li>
<li>The cleaned bones are placed one mouse at a time in the mortar. Two ml of modified PBS is added to the mortar and the bones are crushed using a pestle. It is important to first fragment the bones with 10 to and fro movements of the pestle and then pulverize them with circular rotatory movements. I usually do 50 clockwise, then 50 counterclockwise movements and so on until the material left in the mortar is white in color and the modified PBS added to the material remains white or transparent.</li>
<li>After every set of 50 circular crushing movements, we transfer the fluid from the mortar to a 70 uM filter placed on top of a 50 ml conical tube. Do not forget to add 2 ml of PBS to the mortar after every transfer to the filter. You do not want to try and pulverize dry bones or leave them dry for any length of time as this will lead to apoptosis of cells.</li>
<li>Once the material in the mortar becomes white, the material in the filter is transferred to the mortar and the crushing is again performed. This ensures that the cells in the filter are not wasted. Usually it takes one or two sets of circular rotatory movements to again make the material in the mortar white in color at which time the extraction of cells is complete.</li>
<li>The solution in the conical tube is now centrifuged (please remember to keep the cells from the WT and KO mouse separate. Hence you will have two different tubes (one with WT cells, the other with KO cells). Centrifuge at 1200 rpm for 10 minutes.</li>
<li>Aspirate off the supernatant using separate aspirator pipettes for each tube. Add 2 cc of ACK lysis buffer to each pellet. This lysis buffer should be sterile. Incubate on ice for 5 minutes.</li>
<li>Add 8 cc of modified PBS to each tube. Take 10 ul from each tube and pipette into two separate wells in a 96 well plate. Place the tubes in the centrifuge and spin them at 1200 rpm for 10 minutes.</li>
<li>While the tubes are spinning, add 30 ul of tryphan blue dye and 60 ul of modified PBS to each well of the 96 well plate that carries the cells of interest.</li>
<li>Add 10 ul of this mixture (please mix well by pipetting up and down) to a hemocytometer and count the cells in the four outer squares.</li>
<li>The cell count in each sample per ml is the number of cells thus counted divided by 4 and multiplied by 100 000.</li>
<li>Once the centrifugation is complete, remove the tubes from the centrifuge and resuspend the cells in PBS without calcium, magnesium or L glutamine at a concentration of 20 million cells per ml. You will likely have about 5 ml of volume for the WT and 5 ml of volume for the KO animal. From this step onward we will be using PBS, not modified PBS for the protocol.</li>
<li>To each tube add DiI, a cell staining dye, to achieve a final concentration of 5uM. Vortex immediately for 10 seconds. Incubate at 37 degrees for 10 minutes and avoid exposure to light.</li>
<li>Fill the tubes with PBS and spin for 10 minutes at 1200 rpm.</li>
<li>After centrifugation, aspirate the supernatant and resuspend the cells in PBS at 40 million cells per ml.</li>
<li>Load 500 ul into 1 cc syringes with a 27G needle (5 syringes for the WT mouse and 5 syringes for the KO mice). It may be prudent to prepare 6 syringes for each cohort. Having one extra can help as tail vein injections which we are going to do next can be tricky.</li>
<li>Cover the syringes with foil in order to keep the DiI from disintegrating.</li>
<li>Inject 500 ul of the cell mixture into each recipient animal; (5 for each cohort).</li>
<li>These animals need to be irradiated with 9 Gy of irradiation 4-24 hours prior to injection.</li>
<li>48 hours after injection, harvest bone marrow from the hind limbs of each recipient animal.</li>
<li>Process as described above for the donors. Resuspend the cells from each animal in 1 cc of PBS. Obtain a cell count using a hemacytometer.</li>
<li>Read on the LSR II flow cytometer from BD or an equivalent instrument. The result can be expressed as the percentage or absolute number of cells in the analyzed bone marrow which are DiI positive.</li>
</ol>

<ol>
	<li>Adams, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+engraftment+at+the+endosteal+niche+is+specified+by+the+calcium-sensing+receptor.">Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor.</a> Nature. 439 (7076), 599-603 (2006).</li><li>Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+hematopoiesis.">Chemokines in hematopoiesis.</a> Curr Opin Hematol. 15 (1), 49-58 (2008).</li></ol>

Homing is the process whereby bone marrow cells including stem cells, progenitor cells and differentiated cells, their way into the bone marrow after being injected into the blood stream or bone marrow cavity of mice. As is obvious from the definition, a scientist may chose to study the homing of hematopoietic stem cells, progenitor cells or differentiated cells identified by immunophenotyping and isolated by cell sorting. Typically scientists inject bone marrow cells depleted for lineage markers and hence enriched for progenitor and stem cells.
The dose of the cells of interest injected is a variable in this experiment. The number of cells can vary from 500 000 per recipient mouse to 20 million per recipient mouse. The more cells one can inject the easier the detection in the bone marrow. Cell number availability may limit the number injected. Sorting 20 million LKS SLAM cells is extremely difficult perhaps even impractical with the current technologies in most laboratory settings, in which case one might decide to use a number of cells towards the lower end of the range given above.
The hose animal in which homing is being studied can also be varied. This recipient animal may be a WT C57BL/6J mouse which has been lethally irradiated with 9 Grays of radiation as we did in this experiment, a WT C57BL/6J mouse or a W/Wv mouse which has not been irradiated. Whether the mouse is irradiated or not depends on the question the experimenter is trying to answer. If one is interested in studying homing in a setting where a large amount of cytokines is being released and vascular leak is being induced, an irradiated WT mouse is an appropriate choice. A smaller number of cells (500 000) may be injected into a WT mouse which has not been irradiated so that the stem cells can home to a small number of unoccupied niches without the above confounders.
W/Wv mice are deficient in c-KIT receptor function and can engraft stem cells without any preconditioning. This allows us to study the homing of stem cells in the absence of vascular injury or cytokine release caused by radiation. The logistic problem with the use of these mice is that it is difficult to obtain adequate numbers of WWv mice to serve as recipients due to breeding colony size concerns.
In brief, although we describe a standard homing protocol, the variations used depend upon the question to be answered and must be determined during the design of the experiment.

<table><tbody><tr><td>DPBS (Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Mediatech, Inc.</td><td>21-031-CV</td><td></td></tr><tr><td>Sterile Fetal Bovine Serum</td><td>Valley Biomedical, Inc.</td><td>BS3033</td><td></td></tr><tr><td>0.5M EDTA (pH 8.0)</td><td>Boston BioProducts</td><td>BM-150</td><td></td></tr><tr><td>Tryphan Blue solution</td><td>Mediatech, Inc.</td><td>25-900-CI</td><td></td></tr><tr><td>ACK Lysing Buffer</td><td>Lonza Inc.</td><td>10-548E</td><td></td></tr><tr><td>Isopropranolol U.S.P.</td><td>Denison Pharmaceuticals, Inc</td><td></td><td></td></tr><tr><td>Vybrant DiD cell-labelling solution</td><td>Invitrogen</td><td>V22887</td><td></td></tr><tr><td>Vybrant DiI cell-labelling solution</td><td>Invitrogen</td><td>V22885</td><td></td></tr></tbody></table>

homing of hematopoietic cells to the bone marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

Watch this Scientific Journal Video about Homing of Hematopoietic Cells to the Bone Marrow at JoVE.com

Homing of Hematopoietic Cells to the Bone Marrow

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

homing-of-hematopoietic-cells-to-the-bone-marrow

Nanyang Technological University

Research

JoVE Journal

Biology

29.4K Views.  MGH - Massachusetts General Hospital. This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Video: Homing of Hematopoietic Cells to the Bone Marrow

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Developmental Biology

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

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) functions1,2. 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 progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. 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 &#34;niche&#34; 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.

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

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.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Immunology and Infection

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex extracellular matrix and various cell types, such as mesenchymal stem cells, osteoblasts, and endothelial cells, is deeply involved in hematopoietic stem cell regulation through direct cell-cell interactions, as well as cytokine production. To closely mimic this in vivo structure and conduct experiments reflecting the responses of the human bone marrow, several 3D models have been created based on biomaterials, relying primarily on primary stromal cells. Here, a protocol is described to obtain a minimal and standardized system that is easy to set up and provides features of bone marrow-like structure, which combines different cell populations including endothelial cells, and reflects the heterogeneity of in vivo bone marrow tissue. This 3D bone marrow-like structure-assembled using calcium phosphate-based particles and human cell lines, representative of the bone marrow microenvironment-allows the monitoring of a wide variety of biological processes by combining or replacing different primary cell populations within the system. The final 3D structures can then either be harvested for image analysis after fixation, paraffin-embedding, and histological/immunohistochemical staining for cell localization within the system, or dissociated to collect each cellular component for molecular or functional characterization.

Here, we describe an easy-to-implement, standardized, microphysiological system that reflects the complexity of the human bone marrow's in vivo structure, providing a pertinent model to finely study a broad range of normal and pathological events.

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex ...

Hematopoiesis

The process of blood cell formation is called hematopoiesis. Hematopoiesis starts early during development, on the seventh day of embryogenesis. This phase of hematopoiesis is called the primitive wave, wherein the extraembryonic yolk sac allows the production of erythroid cells and endothelial cells from a common precursor called hemangioblast. The erythroid cells provide oxygen to support the growth of the rapidly dividing embryo. Hemangioblasts later develop into hematopoietic stem cells or HSCs that can produce all types of blood cells due to their multipotent nature. The HSCs then migrate to the liver during the fetal development stage. In adults, hematopoiesis occurs in the bone marrow of organisms.
Inside the bone marrow, there are two populations of HSCs: dormant or quiescent HSCs and the active or primed HSCs. Dormant HSCs are non-proliferative and remain attached to the inner surface of the bone. On the other hand, active HSCs rapidly proliferate in response to vascular endothelial growth factors and are present in the central marrow region.
HSCs migrate from the bone marrow into the circulating blood to reach the different target tissues. During this migration, HSCs lose cell adherence molecules and chemokine receptor CXCR4. HSCs may return to the bone marrow or reach the target tissue by the process called HSC homing. Homing to bone marrow is essential to regulate HSC homeostasis and maintain the HSC pool through proliferation. During homing, HSCs gain cell adherence molecules and chemokine receptor CXCR4 that help them bind and anchor the target tissue following chemokine gradients.&#160;

The process of blood cell formation is called hematopoiesis. Hematopoiesis starts early during development, on the seventh day of embryogenesis. This ...

Education

JoVE Core

Cell Biology

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

KEY TERMS AND CONCEPTS

Multipotency of Hematopoietic Stem Cells

The hematopoietic stem cells or HSCs are multipotent, meaning they can differentiate and give rise to all blood and immune cells. HSCs are maintained in the quiescent stage until an external stimulus initiates their differentiation. The multipotent HSCs exist as two heterogeneous populations, long-term repopulating cells (LTRC) and short-term repopulating cells (STRC). The two HSC populations have different surface markers or receptors and are classified based on quiescence and long-term renewal capacity.
The long-term HSCs rarely divide as they maintain long gaps between successive cell divisions while keeping their metabolic activity to the bare minimum. Thus, the LTRCs undergo a few rounds of symmetric cell divisions before entering the state of dormancy. This allows them to expand the number of HSCs and maintain a stem cell pool before exhausting their regenerative and self-renewal potential.
LTRCs produce the more active multipotent short-term repopulating cells (STRC) in response to external stimuli. STRCs undergo fewer self-renewal divisions than the LTRCs and differentiate into specific blood or immune cells. LTRCs and STRCs find application in engraftment and therapeutic studies. The LTRCs can sustain hematopoiesis for upto four months upon transplantation into the recipients, while transplantation of STRCs supports hematopoiesis for only a few weeks. A delicate balance between dormancy and differentiation stimuli maintains the HSC population for the long run. A slow or non-responsive quiescent HSCs leave the body devoid of differentiated blood cells such as erythrocytes, phagocytes, and lymphocytes which are necessary for transporting nutrients or providing immune surveillance. In contrast, highly active HSCs exhaust the stem cell population making them unavailable to renew, repair, or replace blood cells lost due to an injury or infection.

The hematopoietic stem cells or HSCs are multipotent, meaning they can differentiate and give rise to all blood and immune cells. HSCs are maintained in ...

Homing of Hematopoietic Cells to the Bone Marrow

Summary

Explore More Videos

Chapters in this video

Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone

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

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Tracking Mouse Bone Marrow Monocytes In Vivo

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts

Hematopoiesis

Multipotency of Hematopoietic Stem Cells