Introduction
Human mesenchymal stem cells (hMSCs) from marrow reside
in situ as pericytes that are hypothesized to function as sentinels to guard against self-surveillance by T-cells at sites of tissue damage [
1]. The local titers of hMSCs depend on the vascular density at that site and on other factors. Although hMSCs were first thought to function as the source for cellular replacement therapies, their immuno-modulatory and trophic activities have the potential for profound therapeutic impact in diseases associated with sustained inflammation. By providing additional hMSCs through systemic routes, both immuno-regulatory and regenerative trophic activities at sites of inflammation and tissue damage can be enhanced [
2].
hMSCs are non-hematopoietic, multi-potent progenitor cells, which have the ability to influence immune effector cell development, maturation and function as well as allo-reactive T-cell responses through the production of bioactive cytokines and proteins [
3]. The designation of hMSCs is based upon extensive immunophenotyping using surface antigens and ability to function in
in vitro models [
4]. MSCs are immuno-modulatory and express no MHC class II, making hMSCs a viable therapeutic across tissue typing [
5,
6]. hMSCs produce large quantities of bioactive factors which provide molecular signatures for the pathway and activity status of the responding cells [
7,
8]. These bioactive factors are anti-scarring, angiogenic, anti-apoptotic and regenerative (i.e., mitotic for host-derived progenitor cells). As evidence of the profound effect of hMSCs on the immune system, our colleagues and others have reported that hMSCs are well tolerated and therapeutically active in immuno-competent rodent models of multiple sclerosis and stroke [
9‐
11]. Thus, xenogenic hMSCs repress host immunological surveillance in rodents while at the same time producing reparative growth factors.
An important issue in hMSC biology has been understanding the significant variability among cell preparations. Models need to be developed which not only show the relative unique therapeutic application of hMSCs, but also measure
in vivo function (i.e., therapeutic potency). Acute (short-term)
in vivo models of inflammation have the potential to provide vehicles for hMSC efficacy assessment. Although chronic (long-terms) models provide valid options for study, our focus was on an
in vivo model which would provide a quick answer of hMSC activity, with the clinical and therapeutic applications in mind. Culture-expanded hMSCs increase in size with each passage and thus, on a size basis alone are observed to lodge in the lungs in substantial numbers when given intravenously [
12]. hMSCs have the potential to provide a local source of trophic factors in the pulmonary environment, which may result in changes in lung inflammation [
13]. This circumstance allows us to determine the effects of hMSCs in the initiation of inflammatory lung diseases and to establish the criteria for efficacy of different donor hMSCs on early stages of asthma in rodent models.
Acute bronchial asthma has been characterized by allergic airway inflammation, which induces both cytological as well as histological changes in the airway structure over time [
14]. The pathogenic characteristics of allergic asthma are associated with airway inflammation and infiltration of mast cells, basophils, eosinophils, monocytes and T helper type 2 lymphocytes, along with the production of isotype-specific immunoglobulin E (IgE) [
15,
16]. Several animal models have been developed to model human airway disease associated with acute asthma, which have the capacity to mimic the histological and pathologic changes in the lung. A commonly used model to study airway inflammation
in vivo involves primary sensitization with ovalbumin (OVA) followed by daily intranasal challenge with the antigen to generate airway inflammation mimicking the acute asthma exacerbation. This short-term model can provide the basis for studying the trophic impact of lodged hMSCs on development of lung inflammation associated with acute asthma challenge and the potential benefit of hMSCs in circumventing acute asthmatic inflammatory disease.
The results of these studies provide the foundation for understanding the role of hMSCs in altering inflammatory processes in vivo and provide support for the utilization of the short-term acute asthma model as a validation tool for hMSC efficacy and function in vivo.
Discussion
hMSCs have the unique capacity to be both regenerative and serve as conduits of mediators that can immuno-modulate in situations of inflammation. Intravenously injected hMSCs localize in the lung [
13] prior to disseminating into the peripheral tissues. Asthma is an inflammatory airway disease characterized by T-cell hyper-reactivity, scarring and remodeling. Since hMSCs have the capacity to inhibit scarring and suppress T-cell activity, we investigated the potential of using hMSCs to reverse airway inflammation in the murine ovalbumin model of acute asthma. Our data show for the first time that hMSCs are well tolerated in the murine model of acute asthma, suggesting that hMSCs can favorably change the outcome of asthmatic inflammation without the pathology associated with cross-species application. Further, our data show that hMSCs given after the induction of airway disease dramatically reverse the airway inflammation associated with the ovalbumin model of acute asthma. Additionally, the short-term nature of the acute asthma model and the
in vivo responsiveness to hMSCs suggest that the acute model can be used to measure hMSC effectiveness
in vivo with the correlation of the standard cube score with percent decrease in lung cell recruitment in response to antigenic challenge. The ceramic cube assay measures osteo-differentiation as a measure of hMSC multipotency and has, historically served as the gold standard. The correlation between immunomodulatory activities
in vivo provides an alternative means of evaluating
in vivo potency and efficacy. This is consistent with the observations that the effect of MSCs via paracrine mechanisms or direct interaction with immune cells, do not depend on cell engraftment and differentiation [
35,
36]. Future studies will include hMSC dose-response and different modes of administration.
Adult hMSCs isolated from bone marrow are able to differentiate in culture into a number of mesenchymal phenotypes including those that form bone, cartilage, muscle, fat and other connective tissues [
23]. Originally, it has been suggested that hMSCs are responsible for the normal turnover and maintenance of adult mesenchymal tissues. More recently, hMSCs have been shown to reside in a number of tissues as pericytes, suggesting that they can have a major impact on focal injuries [
1,
2,
41]. If hMSC are capable of impacting the local milieu, they could be used therapeutically as allogeneic sources of repair
in vivo[
42]. The therapeutic implication of hMSCs is based upon the observation that culture-expanded hMSCs have no detectable MHC class II cell surface markers or co-stimulator molecules [
4], suggesting that the hMSCs evade immune surveillance by the host. In our model, hMSCs induce white blood cell recruitment in the acute model when the animals lack the inflammation associated with the secondary challenge with ovalbumin. Although the BAL differential and the total cell counts were altered by hMSCs in the acute control, no detectable changes in lung histology are detected, suggesting the absence of adverse response of the host tissue to the hMSCs in the saline-challenged acute model.
hMSCs were first used to supplement bone marrow transplantations because hMSCs were assumed to home back to the bone marrow stroma and have the potential to efficiently prefabricate the injured marrow stroma for human stem cell engraftment and subsequent hematopoietic lineage functions [
10]. Early successful clinical trials supported the idea that culture-expanded hMSCs were, indeed, capable of promoting successful engraftment of hematopoietic progenitors and their production of circulating mature blood cells efficiently and safely [
43,
44]. Allogeneic engraftment has also been used to treat gene defects [
45,
46]. In these cases, culture-expanded hMSCs from the allo-donor were used to supplement the bone marrow transplantation of host with defective genotypes with the assumption that the hMSCs homed to marrow and re-established the stroma to enhance allogeneic engraftment. For example, Horwitz and colleagues reported improvement in six children with osteogenesis imperfecta treated with allogeneic bone marrow transplantation [
45]. These studies used allogeneic hMSCs with no adverse events. In studies using the bleomycin model, administration of hMSCs into mice immediately after exposure to bleomycin was associated with a significant reduction in inflammation and collagen deposition associated with the lung disease [
47,
48]. In these studies, the rates of engraftment were undetectable or at the limits of detection. The implication is that the mechanism of the hMSC effect and improvement in the bleomycin- induced inflammation was not particularly due to stem cell engraftment of the injured tissue, but to the effects of paracrine secretion of growth factors and cytokines which stimulate repair.
hMSCs have been shown to effectively shut down graft versus host disease (GvDH), a T -cell mismatched immune-mediated disease [
49]. Osiris Therapeutics
http://www.osiristx.com has documented that during a study of compassionate use of adult marrow-derived culture-expanded allogeneic hMSCs in children with steroid resistant GvHD, 7 out of 12 had complete remission of GvHD at one month and 95% were alive at 6 months. In addition, 9 out of 12 had complete recovery from their gastrointestinal GvHD, and the remaining 3 had their severity reduced to Grade I gastrointestinal GvHD. The allogeneic MSCs in these studies, even with the multiple infusions, induced no adverse events
http://www.osiristx.com. This is consistent with our observations using hMSCs in the saline sensitized, saline-challenged control mice. These mice had no change in lung pathology and minimal change in inflammatory status as defined by BAL differential. These and other studies established that isolation and culture expansion is safe with clinical benefit from the intravenous delivery of allogeneic hMSCs. These observations suggest that the trophic effects of hMSCs played a profound role in the observed therapeutic benefit in all of the above studies.
In terms of a potential therapeutic, our data show that hMSCs are effective at attenuating or reversing the inflammation and pathology associated with acute asthma in the ovalbumin murine model of this airway disease. The acute model of murine asthma generated increased pulmonary inflammation and histology consistent with increased mucus production and eosinophils. hMSC treatment of acute asthma mice resulted in decreased airway inflammation as determined by BAL. Histologically, the airways showed signs of decreased epithelial lining thickening and mucus hyper-production. hMSCs in culture secrete a variety of molecules, both bioactive and extracellular matrix, in response to their local environment and their activity status [
7,
8,
50]. In our studies, not only did hMSC in children decrease the pathology and inflammation in the acute murine asthma, but there was a significant decrease in lung IFNγ levels consistent with decreased immune activation. Systemically, administration of hMSCs increased IFNγ, while at the same time decreased IL-1β. These studies suggest a mechanism by which IFNγ/IL-1β levels may alter the host inflammatory response in an allergic setting through altering systemic cytokines. Unlike other models [
35], we did not detect IL-10, IL-5, IL-4 or IL-13 in the BAL fluid from our model, which may be due to tissue localization instead of soluble secretion into the BAL fluid. The differences may also be due to the model of selection. This is the focus of on-going research and future manuscripts. In addition to understanding how hMSCs regulate
in vivo systemic and localized IFNγ/IL-1β levels, our focus will also include factors secreted by the hMSCs or by the host in response to MSCs, which alter the course of asthma. Some possible candidates include TGS-6, SDF-1, MPC-3 and Hif-1α [
51,
52].
Aerosolized corticosteroids are the standard anti-inflammatory medication for asthmatics and are effective in most cases. However, a substantial number of asthmatics remain symptomatic even with the addition of steroid-saving long-acting beta[
2]-adrenoreceptor agonists [
53]. Variability in immune challenge and response to therapy makes asthma a difficult disease to monitor and manage. Further, long term treatment of patients with systemic corticosteroids for severe asthma raises concerns due to the secondary effects of the therapeutic. New treatment options targeting the pathophysiologic events causing development and persistence of asthma are desired for these patients [
54]. The presentation of hMSCs limits the field of damage or injury and inhibits fibrosis or scarring at sites of injury in the ovalbumin model of asthma. The infused hMSCs secrete immuno-modulatory agents which deactivate T-cell surveillance and chronic inflammatory processes [
8]; thus, allogeneic or xenogeneic MSCs can be therapeutically effective. The data presented in this manuscript implicate the trophic products of hMSCs in attenuating inflammation in the acute asthma murine model. Additionally, the acute asthma model of inflammation may be a viable option for gauging hMSC efficacy and function
in vivo in conjuction with
in vitro assays such as the ceramic cube model.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TB directed, analyzed and planned all of the studies. MN carried out the studies and helped with the methods. DL isolated the hMSCs, did the cube scores and provided information on the technology associated with the hMSCs. AC provided insight on the hMSCs impact on the asthma model and the identification of the potential for hMSC efficacy testing. AC laboratory provided the support for the stem cell production, validation and application. All authors read and approved the final manuscript.