Original article
Troubleshooting: Quantification of mobilization of progenitor cell subsets from bone marrow in vivo

https://doi.org/10.1016/j.vascn.2010.01.013Get rights and content

Abstract

Introduction: The molecular mechanisms that control the mobilization of specific stem cell subsets from the bone marrow are currently being intensely investigated. It is anticipated that boosting the mobilization of these stem cells via pharmacological intervention will not only produce more effective strategies for bone marrow transplant patients, but also provide novel therapeutic approaches for tissue regeneration. Methods: Measurement of stem cell mobilization by sampling peripheral blood is problematic because it is technically difficult to accurately determine absolute numbers of rare progenitor cells by blood sampling. Furthermore a rise in progenitors may be caused by release of stem cells from tissues other than the bone marrow (e.g. spleen and adipose), or indeed an inhibition of stem cell homing back to the bone marrow or other tissues. Finally it is not possible to distinguish whether the pharmacological agent is acting directly at the level of the bone marrow or mobilizing progenitors by a distinct indirect mechanism. To resolve these problems, we have developed a technique that allows perfusion of the vasculature of the hind limb bone marrow in situ in mice. In this system, the femoral artery and vein are cannulated in situ such that the femur and tibia bone marrow are perfused in isolation under anaesthesia. As such, pharmacological agents can be administered directly into the bone marrow vasculature. Mobilized cells are then collected via the femoral vein and colony assays performed in defined growth media to allow identification of haematopoietic, endothelial, and mesenchymal progenitor cells. We have used this system to determine the ability of a CXCR4 antagonist to mobilize these distinct types of progenitor cells from the bone marrow of mice pre-conditioned with either G-CSF or VEGF. Results and conclusion: This isolated hind limb perfusion system has allowed comparisons to be made between cytokines (G-CSF and VEGF) that act chronically, either alone or in combination with agents that act acutely on the bone marrow (CXCR4 antagonist) on their ability to directly mobilize specific populations of stem cells. Data obtained therefore gives a more accurate understanding of the efficacy of different mobilizing strategies compared to peripheral blood analysis.

Introduction

It is keenly anticipated that the utilization of stem cells will offer unique opportunities to restore function to injured tissues in the expanding field of regenerative medicine. However, medical procedures have successfully used adult stem cells harvested from the bone marrow to reconstitute the haematopoietic bone marrow niche in leukaemia patients undergoing bone marrow transplants after chemo/radiotherapy for more than 50 years (Thomas et al., 1957, To et al., 1997). Haematopoietic stem/progenitor cell (HSC/HPC) replacement has subsequently been refined through the use of pharmacological agents to mobilize stem cells out of their bone marrow niche into peripheral blood. An example is the prolonged administration of granulocyte-colony stimulating factor (G-CSF) which has facilitated HPC harvesting procedures (Ng-Cashin and Shen, 2004, Ringden et al., 2000). Mobilized HSCs/HPCs using this myeloid cytokine are now the favoured source of transplantable cells to reconstitute a patient's bone marrow and recover haematopoiesis following chemotherapy because they reconstitute the bone marrow more rapidly and provide for a lower long-term relapse rate compared to directly harvested stem cells (Ng-Cashin and Shen, 2004, Ringden et al., 2000). Despite this advancement, it is of note that in 20% of patients treated with G-CSF for bone marrow transplants, insufficient numbers of HSCs/HPCs are mobilized to perform a bone marrow transplant (Roberts et al., 1995, Cashen et al., 2004). This insufficiency may be overcome in the future with novel classes of mobilizing agents such as those that maximally mobilize cells within 1–2 h. Examples include C-X-C motif (CXC) chemokines that activate stem cell migration directly (e.g. CXCL2 and CXCL8), and CXC receptor 4 (CXCR4) antagonists (e.g. AMD3100) that disrupt the CXCR4/SDF-1α cellular retention axis within the bone marrow, or strategies that combine daily G-CSF administration with one of these acutely acting agents (Pelus and Fukada, 2008, Broxmeyer et al., 2005, Martin et al., 2006). However, to achieve more efficacious stem cell mobilizing regimens, direct measurements will need to be undertaken to compare the ability of these prospective agents to cause stem cell mobilization from the bone marrow.

It is now appreciated that the adult bone marrow also contains other stem cell types, including endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and epithelial progenitor cells (Asahara et al., 1997; Pittenger et al., 1999, Wong et al., 2009, Gomperts et al., 2006, Krause, 2008). The molecular mechanisms by which these cells egress from the bone marrow and the physiological role of these distinct stem and progenitor cell subsets are currently the subject of an intense research effort. However, as yet, there have been no reports of reproducible, efficacious strategies for mobilizing these distinct stem and progenitor cells from the bone marrow.

Mobilized EPCs may facilitate post-natal neovascularisation in adults and they might therefore be of therapeutic value in diseases where ischemic tissue requires reperfusion around existing occluded vessels in order to regain organ function (Asahara et al., 1997; Nolan et al., 2007, Yoder et al., 2007). Recent elegant experiments using multiple model systems to evaluate angiogenesis in mice confirm that there is a significant bone marrow stem and progenitor cell input into neovascularisation, and that this is dependent on the nature of the lesion; whereby the more severe the ischemic signal, the greater the contribution of the bone marrow (Madlambayan et al., 2009). Thus far, it is known that EPCs can be mobilized from the bone marrow by a variety of stimuli, including cytokines: G-CSF, vascular endothelial growth factor (VEGF); CXCR4 antagonists; chemokines: CXCL2, CXCL8; nitric oxide (NO), and angiopoietin (Takahashi et al., 1999, Pitchford et al., 2009, Shepherd et al., 2006, Ozuyaman et al., 2005, Moore et al., 2001, Hristov et al., 2007). However, there is little, if any data, directly comparing the efficacy of these mediators in mobilizing EPCs as possible agents to induce therapeutic angiogenesis.

MSCs were first characterised by Pittenger et al. (1999) as cells that were adherent to plastic surfaces and derived from adult bone marrow, with the ability to differentiate into adipocytes, osteocytes, and chondrocytes. There is now evidence that bone marrow-derived MSCs can also differentiate into skeletal muscle, smooth muscle, cardiac myocytes, and neurones and they therefore show a multipotency that could be harnessed therapeutically to restore damaged tissues (Roufosse, Direkze, Otto & Wright, 2004). Attempts to mobilize MSCs from the bone marrow with G-CSF treatment (a cytokine that mobilizes both HPCs and EPCs) have been largely disappointing (Lazarus et al., 1997, Wexler et al., 2003, Roufosse et al., 2004, Pitchford et al., 2009). Nevertheless, the detection of circulating MSCs in response to tissue injury suggests MSCs can be mobilized from their niches upon appropriate signals and that they may therefore target damaged tissue (Mansilla et al., 2006, Wang et al., 2006). We have recently reported direct mobilization of MSCs from the bone marrow after a unique combination of VEGF pre-conditioning followed by CXCR4 antagonist administration, which suggests that particular molecular mechanisms do exist and can be exploited to boost MSC egress from the bone marrow (Pitchford et al., 2009).

These distinct progenitor cell types may therefore be involved in organ regeneration following injury. A greater understanding of the molecular mechanisms that induce or control their retention and egress from the bone marrow will help deliver in an expanding area of pharmacology to boost stem cell mobilization as a novel therapeutic approach in regenerative medicine. Methodology and techniques need to be developed and implemented to make the necessary direct comparisons of the potency of different pharmacological agents in their ability to mobilize stem cells, and to illicit differential and optimal mobilization of distinct stem cell subsets, dependent on the disease. As we will discuss below, this is sometimes very difficult to conduct purely by peripheral blood analysis. There is therefore a requirement for more refined procedures that measure stem cell egress direct from the bone marrow during pre-clinical experimentation.

Reports examining the ability of agents to mobilize stem cells rely upon analysis of circulating levels of progenitor cells, often at a single time point. Such techniques are further hindered by the fact that the mobilizing agent of interest is often administered systemically (rather than directly infused into the vasculature of the bone marrow), with no appreciation of how integrative the body's response that leads to stem cell mobilization may be. Whilst such approaches appear satisfactory for the simple goal of detection of HPCs and EPCs (Broxmeyer et al., 2005, Shepherd et al., 2006), this procedure has fundamental limitations.

The adult body contains many stem cell niches other than the bone marrow from which stem cells can be mobilized. Notable examples include: myocardial tissue, adipose tissue, spleen, liver, and lungs (Wright et al., 2001, Han et al., 2010, Heeschen et al., 2003, Pereira et al., 1995, Prockop, 1997). Therefore, detection of peripherally circulating stem cells after administration of ‘mobilizing’ agents may include circulating stem and progenitor cells mobilized from multiple distinct niches. Thus, factors that mobilize progenitor cells via established mechanisms from the bone marrow may also regulate the mobilization of tissue specific stem cells via poorly understood pathways. Furthermore, identification of a particular stem or progenitor cell subset (e.g. HPCs, EPCs, and MSCs) may actually include a heterogeneous population with subtle differences in characteristics which are a result of their tissue origin. This may affect their functionality, their differentiation potential, and also their ability to traffic to subsequent tissues of interest if different from where their original niche resides (Jaiswal and Weissman, 2010, da Silva Meirelles et al., 2009).

Once mobilized, stem cells can be rapidly recruited to the site of injury, home back to the bone marrow (within minutes) or redistribute to other tissues with local stem cell niches such as the liver, spleen, and lungs (Kollet et al., 2003, Wright et al., 2001, Hidalgo et al., 2002, Broxmeyer et al., 2005, Heeschen et al., 2003). Thus, the absolute number of cells mobilized remains unknown. This situation is further complicated by reports stating that homing of mobilized stem cells back to bone marrow relies on different molecular mechanisms, depending on the mobilizing regimen used in the first instance (Pelus & Fukada, 2008). For example, CXCL2 mobilized HSCs are less dependent on CXCR4 compared to G-CSF mobilized HSCs, and are instead selectin and integrin dependent (Bonig, Priesly, Oehler & Papayannopoulou, 2007). Homing kinetics are therefore also likely to differ. The overall kinetics of mobilization from the bone marrow cannot therefore be assessed as HPCs continually exit and return to the bone marrow at different rates depending on the molecular mechanism by which the mobilizing agent acts. Quantitative comparisons of the efficacy of different drugs to mobilize stem cells cannot therefore easily be obtained by sampling peripheral blood. Furthermore, diurnal variation of levels of SDF-1α in the bone marrow means that systemic levels vary depending on the time of day/night (Mendez-Ferrer, Lucas, Battista & Frenette, 2008).

Identification of the far less numerous EPCs and MSCs is problematic compared to HPCs. Reported comparisons between the ability of a CXCR4 antagonist and G-CSF to mobilize EPCs revealed very low numbers of detected cells per ml of blood (0.5 and 1 cell/ml respectively) (Shepherd et al., 2006). Further, MSC mobilization to various chemokines or pharmacological agents has been even more elusive to obtain from peripheral blood analysis (Fox, Chamberlain, Ashton & Middleton, 2007). It is therefore also difficult to determine whether a pharmacological agent is acting to differentially mobilize distinct stem cell populations or is acting as a ‘pan’-mobilizer. This is an important point because the ‘unseen’ mobilization of other stem cell subsets, or indeed leukocytes may be deleterious depending on the disease condition.

To better understand how pharmacological agents affect molecular mechanisms involved in the mobilization of stem cells, it is impossible to attribute whether effects are local (e.g. confined to the bone marrow) or systemic if mobilizing agents are only administered remotely. For example, stem cell mobilization can occur via β2 adrenergic stimulation of the sympathetic nervous system (Katayama et al., 2006), under central autonomic control (Denes et al., 2005, Spiegel et al., 2007). Therefore it is possible for some systemically administered compounds to affect neuronal input of the bone marrow. Furthermore, parenteral administration of substances may either act directly on the bone marrow, or instead provide a pro-inflammatory stimulus acting at the focal point of administration that culminates in the secondary release of mediators from remotely activated cells, and these two potential mechanisms are difficult to dissociate (Jones, Pitchford, Lloyd & Rankin, 2010).

Section snippets

Resolution: in situ perfusion of mouse hind limb

We have developed an in situ perfusion model of the murine hind limb to directly assess mobilization in vivo. This system has the advantage of measuring the absolute numbers of progenitor cells mobilized over a defined time period. The number of collected progenitor cells can therefore be correctly attributed to their rate of egress (e.g. mobilization) out of the bone marrow and overcomes the problems of peripheral blood sampling as discussed above. This system also permits the infusion of

Discussion and future directions

Using the in situ perfusion system of the mouse hind limb, we have recently tested the ability of various known mobilizing agents (G-CSF, VEGF, and a CXCR4 antagonist), alone or in combination, and compared their ability to mobilize HPCs, EPCs, or MSCs (Fig. 3A; Pitchford et al., 2009).1

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