Defining human mesenchymal stem cell efficacy in vivo
© Bonfield et al; licensee BioMed Central Ltd. 2010
Received: 29 July 2010
Accepted: 25 October 2010
Published: 25 October 2010
Allogeneic human mesenchymal stem cells (hMSCs) can suppress graft versus host disease (GvHD) and have profound anti-inflammatory and regenerative capacity in stroke, infarct, spinal cord injury, meniscus regeneration, tendinitis, acute renal failure, and heart disease in human and animal models of disease. There is significant clinical hMSC variability in efficacy and the ultimate response in vivo. The challenge in hMSC based therapy is defining the efficacy of hMSC in vivo. Models which may provide insight into hMSC bioactivity in vivo would provide a means to distinguish hMSCs for clinical utility. hMSC function has been described as both regenerative and trophic through the production of bioactive factors. The regenerative component involves the multi-potentiality of hMSC progenitor differentiation. The secreted factors generated by the hMSCs are milieu and injury specific providing unique niches for responses in vivo. These bioactive factors are anti-scarring, angiogenic, anti-apoptotic as well as regenerative. Further, from an immunological standpoint, hMSC's can avoid host immune response, providing xenographic applications. To study the in vivo immuno-regulatory effectiveness of hMSCs, we used the ovalbumin challenge model of acute asthma. This is a quick 3 week in vivo pulmonary inflammation model with readily accessible ways of measuring effectiveness of hMSCs. Our data show that there is a direct correlation between the traditional ceramic cube score to hMSCs attenuation of cellular recruitment due to ovalbumin challenge. The results from these studies verify the in vivo immuno-modulator effectiveness of hMSCs and support the potential use of the ovalbumin model as an in vivo model of hMSC potency and efficacy. Our data also support future directions toward exploring hMSCs as an alternative therapeutic for the treatment of airway inflammation associated with asthma.
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 . 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 .
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 . The designation of hMSCs is based upon extensive immunophenotyping using surface antigens and ability to function in in vitro models . 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 . hMSCs have the potential to provide a local source of trophic factors in the pulmonary environment, which may result in changes in lung inflammation . 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 . 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.
Materials and methods
Balb/c mice were purchased from the Jackson Laboratories (Bar Harbor, Maine) at 5 weeks, allowed to adjust to environment for 1 week. On day 0 (6 weeks of age) mice were sensitized by intra-peritoneal injections (100 μL) of 10 μg of ovalbumin (OVA) emulsified in 1.5 mg of Al (OH)3. On day 14, mice were exposed to 1% wt/vol OVA [17, 18] in sterile saline by aerosolization every day for 5 days. Sham sensitization and sham challenges were carried out with sterile saline. hMSCs were given on either day 14 or day 16 by tail vein injection with 1 × 106 hMSC/mouse in 100 ul of PBS. For each of a minimum of 3 experiments, 4-6 animals were evaluated for the following: saline challenge, saline challenge +hMSC, ovalbumin challenge, ovalbumin challenge+hMSCs. In addition, subsets of saline and ovalbumin mice were given bone marrow derived macrophages (BMDM) as a control for the hMSC. As a positive control for immunosuppression, a subset of the ovalbumin sensitized, ovalbumin challenged mice received dexamethasone at 10 mg/kg. In all cases, the dexamethasone significantly decreased total cell recruitment and eosinophil counts (data not shown) supporting our ability to measure changes in airway inflammation.
Mice were injected subcutaneously with ketamine (80 mg/kg) and xylazine (10 mg/kg). The thoracic cavity was opened and lungs exposed. Bronchoalveolar lavage (BAL) was performed by inserting a cannula through a cut in the trachea into the bronchi and infusing 3 × 1 ml aliquots of warm PBS containing 0.2% lidocaine. The BAL fluid sample was recovered by aspirating the liquid with a syringe. Cells were separated from lavage fluid and differential analysis was evaluated using cytospins and Wright-Giemsa staining. Remaining cells were frozen for future analysis. BAL fluid was frozen at -80°C until assessment for cytokines.
Lungs were either perfused with 10% formalin or snap frozen. Mean viability of lavage cells was > 95% by trypan blue dye exclusion. Animals were assessed for inflammation by BAL and a separate set of animals was evaluated for lung histology using H&E and trichrome staining.
Animals underwent cardiac puncture after completion of lavage and the blood was processed to obtain serum and plasma. Serum cytokines were measured using ELISAs or Luminex technology.
Cells were isolated from the marrow of healthy volunteers as described elsewhere . Briefly, the marrow aspirates were layered on density gradients, and the light cell fraction was retrieved, washed and plated in selected batches of 10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) . Within 14 days the colony-forming units were selectively expanded to near confluency and the cells were then lifted from the dish with trypsin and replated at 4500 cells/cm2 to ensure they maintained active cell division. As determined by other studies, cells cultured using this protocol were homogenous when FAC-Sorted using over 100 different cell surface antibodies . All 7 hMSCs preparations were harvested from culture at passage 2, during log-growth and given intravenously. Just before cells are trypsinized, they are rinsed with Tyrode's salt solution then labeled with the vital dye, Dil at a concentration of 1 ug/ml in Tyrode's for 20 minutes at 37°C. Dil was initially diluted to 1 mg/ml in DMSO followed by 1:1000 diluted in Tyrode incubated for 20 minutes at 37°C. Each experimental run used hMSCs from a different donor; 7 different hMSC preparations were used for these studies.
Bone marrow derived macrophages (BMDM)
BMDM were utilized in a subset of studies (n = 3). Briefly, bone marrow aspirates were obtained from syngeneic mice and cultured in the presence of L929 media as previously defined .
Ceramic Cube Assay
Cube Score Evaluation of hMSCs Derived from Human Marrow
Age of Donor
Cell Yield/Plate (× 106)
Cytokine and Inflammatory Mediator Assessment
BAL and serum differentials were measured in duplicate from each individual mouse (no samples were batched) by cytospins and direct cell counts . BAL level of cytokines (R&D Systems, Minneapolis, MN) were assessed by ELISAs and Luminex multiplex assays as previously described . Inter and intra assay variability was controlled through the use of high and low quality control standards and standard curve slope comparison using Statlia Technology (San Diego, CA). The lower range of detection was between 3-10 pg/ml depending on the cytokine in evaluation.
Group and time point comparisons used repeated measures of analysis of variance (ANOVA) and t-tests. Individual significance levels of 0.05 were used for all tests. The designation of "n" in these studies is based upon the number of experimental times the study was performed. In each experiment, there were 4 groups: saline challenged-no hMSCs, saline challenged-hMSCs, ovalbumin challenged-no hMSCs, ovalbumin challenged-hMSCs. Groups were separated into histology/pathology or bronchoalveolar lavage. Each group of histology/pathology had 3-4 mice. Each group of inflammation had 5-7 mice. Statistics were done based upon the total number of experiments including all of the animals in each of the different groups. In some instances a description identifying "n" versus the numbers of animals in each group is explained for clarity.
The Asthma Models
Localization of Human Mesenchymal Stem Cells
Human Stem Cells in Murine Models: Xenographic Effects
hMSC Effect on Murine Models of Asthma Inflammation
Administration of hMSCs Alters both Local and Systemic Cytokines
Dependence of The hMSC Effect on Existing Inflammation
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  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 . 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. 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 , 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 . 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 . 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 . 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 , 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-adrenoreceptor agonists . 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 . 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 ; 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.
We would like to thank David Carrino for helping in the preparation of this manuscript. We would also like to thank Sung Eun Yang, Brandon Zuchowski and Brian Jennings for the technical help with the asthma model. The funding for this research was graciously provide by the David and Virginia Baldwin Fund, Center for Stem Cell and Regenerative Medicine and the Case Western Reserve Vision Fund.
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