The heart is made by different types of cells. Therefore, optimum cell therapy for cardiac repair may require the association of different cell populations with complementary activities leading to balanced cardiomyogenesis/vasculogenesis. Apart from a recent study reporting the benefit of associative treatment with human MSCs and human c-Kit+ CSCs in an immunosuppressed swine model of myocardial infarction (Williams et al., 2013), combinatory cell therapy has received very little attention. We recently reported the advantage of dual therapy with CSCs and vascular pericytes for harmonized repair of the infarcted heart in mice (Avolio et al., 2015a). Interestingly, the two cell populations exert reciprocally enhancing paracrine activities, which lead to increased proliferation of vascular cells and cardiomyocytes and attraction of endogenous CSCs. These seminal examples of combinatory cell therapy set the basis for fabrication of scaffolds functionalized with multiple cell types.

#### **Stem cells in the youngest heart**

Despite minor attention has been focused on the pediatric heart, a few studies displayed that CSCs are more abundant in the neonatal period and rapidly decrease over time, making myocardial samples from the youngest hearts an optimal source of stem cells for use in reconstructive surgery (Peral et al., 2014). In particular, the histological examination of RV biopsies from CHD patients aged 2–93 days demonstrated that c-Kit-positive CSCs are four-fold more abundant during the first post-natal month (0.4 vs. 0.1% of total cardiac cells at birth and after 1 month, respectively), and a similar reduction was observed also for NKX2.5-positive cells (Amir et al., 2008). Moreover, Mishra et al., compared c-Kit-positive CSCs isolated from right atrial (RA) specimens of CHD patients selected on the basis of 3 different groups of age: neonates (<30 days), infants (1 month to 2 years) and children (2–13 years), and showed that c-Kit-positive CSCs are two- and three-fold higher in neonates than in infants and children respectively, along with a superior differentiation potential (Mishra et al., 2011). Similarly, a comparison of human hearts aged between 9 days to 77 years showed that the number of CSCs isolated per mg of cardiac tissue was more than three-fold higher in the youngest hearts (Tateishi et al., 2007). In addition to these considerations, a study performed in children aged 19 days to 16 years affected by univentricular heart and chronic pressure overload, showed that the number of c-Kit-positive cells in RV of patients is three-fold higher than in cardiac biopsies posttransplantation, suggesting that the pressure overload may lead to an increase in resident CSCs (Rupp et al., 2012a). This latter may represent an adaptive response in CHD children in the attempt to counteract the alterations in cardiac function, although not sufficient to overcome the challenges of severe CHD.

But stem cells resident in neonatal hearts are not only more abundant than the adult counterparts. A study performed by Simpson et al., showed that c-Kit/CD90-positive CSCs generated from neonatal RA samples are also endowed with a superior regenerative ability than CSCs derived from adult samples, when transplanted in a rat model of acute myocardial infarction (Simpson et al., 2012). Authors showed that neonatal CSCs are characterized by higher levels of c-Kit, flk-1 and Islet-1 positive cells in comparison with the adult ones; the younger cells possessed also an increased cardiomyogenic potential both in vitro and in vivo, preventing the formation of the fibrotic scar and improving cardiac functional recovery when transplanted in the animal hearts. Authors supposed that a possible explanation for this augmented regenerative ability of neonatal CSCs might derive from the higher secretion of pro-angiogenic factors, like Vascular Endothelial Growth Factor (VEGF) and Angiogenin, which play a crucial role during the formation of new blood vessels (Simpson et al., 2012).

Besides the above mentioned CSC classes, other cell types have been isolated from the youngest human heart, and may represent a valid option in cell therapy for correction of CHD. Very recently, a population of heart pericytes (HPs) has been identified by the group of B. Péault in myocardial samples from both fetuses at 17–23 weeks of development and post-mortem adults. HPs, recognized as CD34/45-negative and NG2/PDGFR-β/CD146 positive cells, possess a multilineage mesodermal differentiation potential, including limited cardiomyogenic properties both in vitro and in vivo in a mouse model of MI, and marked proangiogenic ability in vitro (Chen et al., 2015). Despite this, the potential of these cells has not been evaluated immediately after birth yet. Instead, our group has recently been involved in the isolation and characterization of a population of cardiac pericytes (CPs) from myocardial leftovers of neonates and children operated for correction of CHD (Avolio et al., 2015b). We originally select CPs as CD34-positive CD31-negative cells, and after expansion CPs express pericyte (NG2/PDGFR-β) along with stemness markers (NANOG, OCT-4, SOX-2), are clonogenic and endowed with ability to differentiate into VSMCs, but not into ECs or cardiomyocytes. We demonstrated that CPs are able to attract endothelial and cardiac stem cells in an in vitro migration assay, and support the network forming ability of ECs in an in vitro angiogenic assay (Avolio et al., 2015b). Last, in the human fetal aorta a population of CD133/CD34/VEGFR-positive vascular progenitor cells has been identified and characterized for its unique vasculogenic and myogenic potential in a mouse model of limb muscle ischemia (Invernici et al., 2007).

#### Preclinical Trials

Since the complex nature of congenital heart defects makes these latter difficult to reproduce in animals, there are no many animal models of CHD, and this represents a significant limit in the translation of new therapies to clinic (Peral et al., 2014; Tarui et al., 2014). Nevertheless, in the last years some efforts and progresses have been done, in particular to reproduce an increased pressure or volume overload of the RV that is a common feature in patients with a HLHS or ToF. Although it is difficult to make an animal model that mimics univentricular hearts, a pressure-overload right-heart model using pulmonary artery banding has been developed in rats (Hoashi et al., 2009) and sheep (Davies et al., 2010), which is useful to evaluate the safety and efficacy of cell transplantation in presence of single ventricular lesions. In these studies, skeletal myoblasts (Hoashi et al., 2009) and cord blood stem cells (Davies et al., 2010) were injected in the animal hearts, determining an improvement of RV function. Also, a first murine model of RV volume overload has been obtained by Reddy et al., by entrapping the pulmonary valve leaflets with sutures (Reddy et al., 2013). In addition, a recent study performed in a piglet ToF model showed the safety and feasibility of intramyocardial administration of human MesP1 (mesodermal posterior 1)-positive /SSEA-1(stage specific embryonic antigen-1)-positive embryonic stem cell-derived cardiac progenitors 4 months after a surgical procedure mimicking the repair of ToF. This latter consisted of an enlargement of the RVOT by a polytetrafluoroethylene patch, the excision of one pulmonary valve leaflet and a pulmonary artery banding. At 3 months followup, animals receiving cell therapy showed some improvements in RV remodeling (reduction of peri-myocyte fibrosis) but unfortunately no significant improvement in RV function compared to control animals (Lambert et al., 2015). Finally, the feasibility and long term safety of autologous UCBMNCs transplanted intramyocardially into the RV of piglet hearts was recently demonstrated (Cantero Peral et al., 2015).

#### Clinical Trials in Children with CHD

So far, major attention has been dedicated to the therapy of pediatric dilated cardiomyopathy (Rupp et al., 2009; Bergmane et al., 2013; Selem et al., 2013), while experience with stem cell therapy in children with severe CHD or acquired heart failure has been limited to single cases and small-size cohorts.

In 2010, Rupp et al., reported a case of cell therapy with intracoronary injection of autologous BMCs in an 11-monthsold boy with HLHS; although this was only an isolated case, the patient outcome was dramatically improved 3 months after cell therapy, with RV ejection fraction (RVEF) increasing from 22 to 44% (Rupp et al., 2010). More recently, intraoperative administration of autologous UCB-stem cells in a 4-months-old baby undergoing a second palliation surgery gave positive results, improving the RVEF from 30 to 50% at 3 months of followup (Burkhart et al., 2015). Again, another study performed in 9 children with severe terminal HF (6 with HF secondary to dilated cardiomyopathy and 3 with CHD) showed the feasibility and safety of intracoronary infusion of autologous BMMNCs, stabilizing patients conditions during the short (3 months) and long term (up to 52 months) follow-up (Rupp et al., 2012b).

The first long term follow-up phase I controlled clinical trial in pediatric patients with CHD, using autologous CSCs, has been concluded only recently (Ishigami et al., 2015). In the TICAP trial (Transcoronary infusion of Cardiac progenitor Cells in patients with single ventricle physiology), autologous CSCs were isolated from 7 children affected by HLHS, aged 5 months to 3 years, and administered via intracoronary delivery 4–5 weeks after surgical palliation. Seven children were selected as control (received palliation surgery without cell therapy). The administration procedure was safe, without any serious adverse event. At 18 months of follow-up, the CSC-treated patients demonstrated an improvement in RVEF from an average baseline value of 47 to 54%, whereas control patients showed little improvement in RVEF, from 47 to 49% (Ishigami et al., 2015). This study gave the first important demonstration of safety and feasibility of autologous CSCs application in young CHD patients. The same group is now carrying out a larger phase II study (Cardiac progenitor Cell infusion to treat univentricular heart disease: PERSEUS), which involves 34 patients randomly assigned to the treatment or control group, to strengthen the positive results collected during the first phase (Tarui et al., 2014).

# Tissue Engineering for CHD correction

Tissue engineering is a promising bioengineering technology that aims at the creation of unique substitutes using threedimensional synthetic or biologic scaffolds seeded with autologous stem cells and differentiated cells, to allow individual patients therapy with regeneration, remodeling and growth potential (Cheema et al., 2012; Smit and Dohmen, 2015). On the one hand, scaffolds serve as a site of cell attachment and new tissue formation. On the other hand, their recolonization by cells after implantation seemingly contributes to the creation of a living tissue perfectly integrated in the recipient's organ. Seeded cells may contribute to the homing of resident cells by secretion of chemoattractant factors.

Tissue engineering has been one of the most promising strategies for the regeneration of impaired tissues. For instance, a tissue engineering approach has been used successfully for the repair of injuries to, or congenital absence of complex organs such as the trachea, esophagus, or skeletal muscle (Macchiarini et al., 2008; Badylak et al., 2012). The first success, in 1994, was the reproduction of tracheal cartilage using tissue engineering techniques (Vacanti et al., 1994). Later, bioengineering techniques have been applied also to the heart. Experience accumulated in adult patients suggests that dispersed stem cells may die soon after implantation in the heart, their therapeutic action being attributable to paracrine factors released during the initial post-transplantation phase. Therefore, this approach does not seem to be advantageous for the repair of complex congenital defects, that frequently requires additional tissue in various forms, such as patches, valves and conduits (Dean et al., 2012; Kalfa and Bacha, 2013; Smit and Dohmen, 2015). Stem cells may work more efficiently if embedded in extracellular matrices, prosthetic grafts and patches to create biological structures that, once implanted in the defective heart, can grow and remodel in a physiologic manner in parallel with cardiac and whole body growth (**Figure 3**) (Bertipaglia et al., 2003; Scholl et al., 2010).

#### Which Characteristics Should an Optimal Bioengineered Graft Have?

The creation of an optimal bioengineered graft relies upon the careful choice of two main protagonists: (1) the biomaterial and (2) the cells that will repopulate the biomaterial graft. Scaffolds used in cardiac surgery should provide structural integrity at the time of repair, while permitting infiltration of cells from native tissue and enabling the development of new vessels and blood supply, which contributes to tissue remodeling. The cell-scaffold constructs are configured to serve as a template to promote development of structures that closely mimic the original tissues (Cheema et al., 2012; Fallahiarezoudar et al., 2015). Interestingly, physiological cyclic strain and shear stress may even accelerate the formation of tissue starting from the cellularized graft (Engelmayr et al., 2006). The final result should be a new tissue composed entirely of the patient's autologous cells, able to remodel in response to physiological changes (Butcher et al., 2011; Dean et al., 2012). Not less important, an advantage of coating the prosthetic materials with stem cells or ECs before transplantation is the prevention of thrombotic complications.

The optimal reparative patch for closure of atrial and ventricular septal defects and RVOT reconstruction, as well as the optimal substitutive valves used in pediatric CHD, ideally should possess the following characteristics (Butcher et al., 2011; Dean et al., 2012; Kalfa and Bacha, 2013; Alsoufi, 2014):

These characteristics are critical especially in newborns, in which corrections should be durable for all the course of a normal lifespan, to avoid repeated risky operations. But to date, the optimal scaffold still does not exist, although many progresses have been done in this direction, and a wide series of biological and synthetic materials are currently under evaluation in the attempt to find the optimized one.

An important factor to consider during the creation of a bioengineered graft is its mechanical behavior. Before proceeding with any preclinical validation, the performance of bioengineered grafts is first evaluated ex vivo. This is done both before and after the seeding of cells, to understand how the cells affect the matrix remodeling in biological grafts. During these tests, the hydrodynamics functions and the durability of the graft are evaluated, both under static conditions and in presence of pulsatile flow. The elasticity and stiffness of the graft are evaluated with both uniaxial and biaxial mechanical tests (Ghanbari et al., 2009).

#### Adaptability of Currently Available Grafts to Tissue Engineering

Some undesirable characteristics of currently available grafts discourage the application in CHD patients, but fortunately progresses have been done and promising solutions are underway. The main advantages and disadvantages of materials used for correction of CHD are recapitulated in **Figure 4**.

One matter of concern is that the toxicity of chemical agents used during manufacture of prostheses might inhibit the functionalization by autologous cells. A coating with bioactive substances has been proposed to overcome the last problem and promote the process of recellularization in vivo Dohmen et al., 2006a; Butcher et al., 2011; Strange et al., 2015. Moreover, the use of glutaraldehyde has been challenged because of concerns about the promotion of calcification. Calcification of grafts, in particular valves, is a major problem since it can cause the failure of the graft and the occurrence of further surgery. The risk of calcification in children is even higher than in adults, as in young patients the mobilization of calcium, due to bone remodeling, accelerates the calcification process. A possible solution is represented by anti-calcification treatments (Butcher et al., 2011; Pok and Jacot, 2011). It remains unknown if calcification may affect cells used for tissue engineering. This risk may be higher for cells, including MSCs, which have the propensity to differentiate into osteocytes under inductive conditions (Ohata and Ozono, 2014). However, it cannot be excluded that other cell types may not only resist to xenograft manufacture agents but also contrast their procalcifying action.

The CorMatrix R ECM (CorMatrix Alpharetta, GA), a patch made of decellularized porcine small intestinal submucosa extracellular matrix (SIS-ECM), displays a lot of potential advantages over other materials currently used in pediatric cardiac surgery, such as ease handling and implantability, abundance, low immunogenicity, high mechanical strength, minimal scar formation, recruitment of host cells, remodeling without calcification and possible growth potential (Pok and Jacot, 2011; Kalfa and Bacha, 2013). CorMatrix patches have been successfully used in congenital heart surgery for pediatric cardiac and vascular reconstructions (Scholl et al., 2010; Quarti et al., 2011; Witt et al., 2013). Recently, our group confirmed the great recellularization properties of the CorMatrix, successfully growing cardiac pericytes—isolated from children affected by CHD—onto CorMatrix patches up to 3 weeks in a bioreactor, finally showing the engraftment and viability of cells (Avolio et al., 2015b).

Recently, scaffolds have been fabricated using synthetic biodegradable polymers, such as polyglicolyc acid (PGA), poly-Llactic acid (PLLA) and polycaprolactone (PCL). On one hand, the advantages of using these polymers are a limitless supply, ease of shaping, and the possibility to combine the concentrations of the polymers to generate scaffolds that meet the compliance specifications of the environment in which they have to be introduced. But on the other hand, the degradation process by hydrolysis will affect the mechanical properties over time. The degradation rates have been estimated in 2–3 months for PGA and 2 and 3 years for PLLA and PCL, respectively. Although some concerns have been reported about the cytotoxicity of the products of the degradation process (Butcher et al., 2011; Dean et al., 2012; Fallahiarezoudar et al., 2015), these materials are currently the most used in tissue-engineering procedures for the construction of autologous grafts made by the patient's own tissue.

#### Methods of Cell Seeding and Growth of Tissues In Vitro

During the generation of a bioengineered graft, not only the selection of the proper cell type and the choice of a physiological culture method are crucial in order to achieve good results, but also cell seeding is one of the key issues. How to seed cells efficiently and uniformly, especially in the inner parts of scaffolds with pores, and with no impairment to the cells, has been one of the major challenges for tissue engineering.

Since from the beginning, static seeding has been the most frequently used because of its simplicity and the ease to be performed within a common cell culture laboratory, since it does not require sophisticated equipment. However, the efficiency of static seeding is always low, and the distribution of the cells throughout the scaffold is not uniform, with the inner part of the scaffold holding few cells, even if excellent biocompatible scaffolds with big pores are used. As a consequence, only the superficial layer of the scaffold will be formed by the regenerated tissue (Dai et al., 2009).

One important factor that limits the penetration of cells inside the scaffold is represented by air in the pores, which explains why static seeding cannot reach satisfactory results in term of scaffold repopulation. Different solutions have been considered to overcome this bottleneck.

In centrifugation seeding, a moderate centrifugal force is applied during the seeding process, to facilitate the penetration of cells in the central part of the scaffold (Godbey et al., 2004; Roh et al., 2007). However, whether survived cells remain functional after centrifugation has still to be determined. Moreover, during centrifugation the orientation of the scaffold cannot be controlled, thus that the overlapping of material pieces does not allow a homogeneous distribution of cells. Instead, the low-pressure seeding uses a vacuum desiccator to create a reduced pressure that draws the air in the materials out by pressure difference, facilitating the penetration of the culture media and cells inside the pores (Torigoe et al., 2007). However, despite the seeding efficiency is increased, the low pressure may affect cell viability and function after seeding, thus additional investigations are required (Dai et al., 2009). In perfusion seeding, a continuous cell suspension perfusion is applied through 3Dscaffold pores using bioreactors to assist in cell infiltration and to aid in nutrition (Dai et al., 2009).

A bioreactor is a device able to ensure the biological and physiological environment of the heart and the circulatory system. Mimicking the physiological mechanical forces, shear stress and blood flow, the bioreactor can allow the construction of engineered scaffolds that will be functional after transplantation into the patients (Carrier et al., 1999; Hecker and Birla, 2007). When cellularized scaffolds are kept in culture for long time, the tissue grows and reaches a 3D-structure, so that the static culture system is not adequate to guarantee the perfusion of culture media uniformly. A solution to this problem comes from the dynamic bioreactors, which can maintain viability of tissue-engineered organs by using both mechanical and biochemical conditioning. Apart the basic requirements of cell cultures—which include the control over the time of dissolved O2 and CO2, pH, temperature and nutrients concentration—a dynamic bioreactor can provide the control of flow waveform and physiological pressure, keeping culture medium under the pulsatile flow that enables the perfusion of the media inside the entire graft and simulates at the same time the in vivo pressure and heart rate (Carrier et al., 1999; Hecker and Birla, 2007).

#### Preclinical Studies and Clinical Application of Tissue Engineered-grafts

Tissue engineering-based grafts have been tested in small and large animal models to assess their safety and feasibility before translation to clinics. So far, BMMNCs and ECs have been frequently selected for preclinical studies of tissue engineering applications, probably due to the relative ease of harvesting BM aspirates and peripheral veins compared, for example, to samples of cardiac source. For the sake of brevity and clarity, main animal studies are summarized in **Table 1**. During these procedures, scaffolds were firstly seeded with cells in vitro, and secondly implanted into the animals. The results obtained with animals encouraged the test in humans.

Positive results have been obtained also from clinical studies involving the application of tissue engineering-based grafts in children affected by CHD, although they are still limited to few reports. In 2001, the transplantation of a tissue engineeringbased graft on a 4-years-old girl with univentricular heart and pulmonary atresia was recorded. Autologous cells were isolated from a peripheral vein and seeded onto a biodegradable tubular scaffold composed by PLLA/PCL reinforced with PGA, designed in order to degrade within 8 weeks. Seven months after implantation, the conduit was not occluded and there was no evidence of aneurysm (Shin'oka et al., 2001). Two years later the same group reported other clinical cases of children with CHD who received a biodegradable tissue engineeringbased graft (PLLA/PCL, designed to be degraded in 3–5 years) seeded with total BMCs or selected BMMNCs. Again, the results were positive, with no stenosis documented in the grafts (Matsumura et al., 2003a). The successful transplantation of a vascular biodegradable conduit (PLLA/PCL reinforced with PGA) seeded with saphenous vein derived cells was recorded in a 12-years-old during the Fontan operation (Naito et al., 2003). Later, the clinical results of the application of tissue engineeringbased grafts seeded with autologous BMCs in CHD patients (25 out of 42 patients recruited were <7 years) were published (Shin'oka et al., 2005). The scaffolds were made by PLLA/PCL and PGA, degradable in 2 years. The results were positive, without thrombosis, stenosis or obstruction of the grafts after a median of 16 months; grafts remained patent and their diameter increased in size (Shin'oka et al., 2005). More recently, a similar study was conducted in patients with single ventricle physiology (17 out of 25 patients recruited were <7 years), in which PLLA/PCL/PGA grafts seeded with autologous BMMNCs were implanted as extracardiac cavopulmonary conduits. Four out of 25 patients presented graft obstruction that required angioplasty, but all the other patients experimented a good recovery without failure of the graft (Hibino et al., 2010).

Despite these first encouraging results, the performance of TEgrafts in CHD children still needs to be investigated, since a main limitation of the studies performed so far is the lack of a long term follow-up. Nevertheless, application of tissue engineeringbased grafts to the correction of CHD is still a new field of study, which will be extensively expanded in the coming future.

# Future Perspectives

The choice of the appropriate cell type to use for cell therapy is of crucial importance to reach the desired therapeutic goal. In the future, cell types able to guarantee on the one hand the regeneration of the missing or lost tissue, and on the other hand the parallel remodeling of the extracellular matrix in order to provide the new generated tissue with the ability to adapt with the anatomy of the growing cardiovascular system, might be privileged; also, combinatory cell products may represent an interesting and safe option. But, while there is space for improvement in the characterization and quality of the cell product, the field of regenerative medicine for treatment of CHD urgently requires tissue engineering solutions, as dispersed cells are not ideal to reconstruct valves and conduits. To date, one major limitation in the use of prosthetic devices is the need of replacement. Research is currently underway to provide biologically compatible solutions. While there is a remarkably growing interest in possibilities offered by cell therapy and tissue engineering, the clinical application in newborns and children is still in its pioneering phase.

Cell therapy products or tissue-engineered products are classified as advanced therapy medicinal products (ATMP) that need to be authorized through centralized procedures. In a tissue-engineered product both components (the scaffold and cell product) must be GMP grade and the combination of the two should be also GMP certified. This includes the manufacture and expansion of cells in the scaffold prior implantation, demonstration of conserved mechanical properties and safety/efficacy in adequate animal models. Therefore, it is likely that the first products to become available for clinical experimentation will be those made by clinical grafts containing cells that have been already tested in clinical trials. In term of safety, it should be kept in mind that CHD is increasingly acknowledged to be associated with genetic polymorphisms. Whether alterations in the genetic program that controls the proper formation of the heart may influence stem cell behavior remains unknown and may represent an additional challenge for the progress of the field.

Advancements in diagnostic screening allow an early diagnosis of CHD and set the basis for guiding the best reconstructive strategy as illustrated in **Figure 5**. In the first scenario, cells are obtained prenatally or at birth with minimally invasive procedures, expanded, incorporated in scaffolds ready for use in definitive surgical correction without need of palliative procedures. Induced pluripotent cells could be ideally generated to this purpose. Alternatively, cord cells have a potential for cases that do not require an immediate intervention at birth. In the second scenario, when diagnosis is made post-natally, stem cells from remnants of palliative surgery could be expanded in vitro and combined with biomaterials using bioengineering techniques; afterwards, the resulting cellularized graft will be surgically implanted into the diseased heart at the occasion of a second open-chest surgery, helping the surgeon in performing the reconstructive procedures required to manage complex CHD. Looking at the best cell type, the use of stem cells from cardiac source may give the advantage of a physiological commitment to originate all the 3 major cell types of the heart: cardiomyocytes, ECs and VSMCs. Additional cells with complementary activities, namely vascular cells and pericytes could be added in a combinatory approach.

In conclusion, the potential of tissue engineering may provide in the next future definitive solutions for correction of CHD in newborns and children, opening new avenues with immense therapeutic benefits.

## Author Contributions

EA: reviewed the literature, drafted the manuscript and prepared the figures; MC: critically revised the manuscript; PM: reviewed

the literature, drafted and critically revised the manuscript. All the authors have approved the final submission of the manuscript.

## Acknowledgments

This work was supported by (1) manufacture scale up of human pericyte progenitor cells for regenerative medicine, MRC

References

Translational Stem Cell Research Grant; (2) Bristol Biomedical Research Unit in Cardiovascular Disease (lead for Regenerative Medicine workpackage), National Institute Health Research Biomedical Research Unit (NIHR BRU); (3) preclinical trial with human pericyte progenitors in a large animal model of myocardial infarction, BHF special project grant; (4) BHF Centre of Regenerative Medicine; and (5) the Sir Jules Thorn award 2014. **Figures 1**, **2**, **5** have been prepared using SERVIER Medical Art.

right ventricular volume overload. Am. J. Physiol. Heart Circ. Physiol. 304, H1314–H1327. doi: 10.1152/ajpheart.00776.2012

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Avolio, Caputo and Madeddu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Intractable diseases treated with intra-bone marrow-bone marrow transplantation

#### *Ming Li 1, Kuquan Guo2 and Susumu Ikehara1 \**

*1 Department of Stem Cell Disorders, Kansai Medical University, Hirakata City, Japan*

*2 Department of Cardiac Surgery, Beijing Institute of Heart, Lung and Blood Vessel Disease, Beijing Anzhen Hospital Affiliated to Capital Medical University, Beijing, China*

#### *Edited by:*

*Atsushi Asakura, University of Minnesota, USA*

*Reviewed by:*

*Hong Zhang, Harvard Medical School, USA Simone Pacini, University of Pisa, Italy*

#### *\*Correspondence:*

*Susumu Ikehara, Department of Stem Cell Disorders, Kansai Medical University, 2-5-1 Shinmachi, Hirakata City, Osaka 570-1010, Japan e-mail<EMAIL_ADDRESS>
Bone marrow transplantation (BMT) is used to treat hematological disorders, autoimmune diseases (ADs) and lymphoid cancers. Intra bone marrow-BMT (IBM-BMT) has been proven to be a powerful strategy for allogeneic BMT due to the rapid hematopoietic recovery and the complete restoration of T cell functions. IBM-BMT not only replaces hematopoietic stem cells (HSCs) but also mesenchymal stromal cells (MSCs). MSCs are multi-potent stem cells that can be isolated from bone marrow (BM), umbilical cord blood (UCB), and adipose tissue. MSCs play an important role in the support of hematopoiesis, and modify and influence the innate and adaptive immune systems. MSCs also differentiate into mesodermal, endodermal and ectodermal lineage cells to repair tissues. This review aims to summarize the functions of BM-derived-MSCs, and the treatment of intractable diseases such as rheumatoid arthritis (RA) and malignant tumors with IBM-BMT.

 MSCs have been shown to differentiate into osteoblasts, adipocytes (Dominici et al., 2006), cardiomyocytes (Makino et al., 1999), and pancreatic islets (Tang et al., 2004). Furthermore, MSCs have the ability to migrate to injured tissue of liver (van Poll et al., 2008) and heart (Yokokawa et al., 2008). Adipose tissue and BM are the most readily available sources of MSCs because they are easy to harvest, and there are no ethical concerns. BM-derived MSCs (BMMSCs) have a higher degree of commitment to differentiate into chondrogenic and osteogenic lineages than adipose tissue-derived MSCs (Gimble et al., 2007), although there appears to be no difference between adipose tissue-derived MSCs and BM MSCs in terms of immunoregulatory functions and support of hematopoiesis (Poloni et al., 2012). On the other hand, BM MSCs modulate the immune response, suppress allogeneic T cell responses, and prevent the development of graft-versus-host disease (GVHD) (English, 2013).

BM transplantation (BMT) is useful for treating hematopoietic disorders, allogeneic BMT also being used to treat autoimmune diseases (ADs) (Nishimura et al., 1994). Intra-bone marrow-bone marrow transplantation (IBM-BMT) has been proven to be the most effective approach to treating allogeneic BMT, since IBM-BMT can replace not only hematopoietic stem cells (HSCs) but also BMMSCs Thus hematopoietic recovery is rapid, and no GVHD develops even if whole BM cells are injected (Kushida et al., 2001; Ikehara, 2003). In this review, we focus on rheumatoid arthritis (RA) and malignant tumors treated with IBM-BMT.

#### **IMMUNOREGULATORY FUNCTIONS OF BMMSCs**

BMMSCs have been reported to have the ability to modify and influence almost all the cells of the innate and adaptive immune systems mediated by BMMSC soluble factors, including IL-6, M-CSF, IL-10, TGFβ, HGF, and PGE2 (Aggarwal and Pittenger, 2005; Beyth et al., 2005; Ramasamy et al., 2007). The adaptive immune system, which is composed of T and B lymphocytes, generates specific immune responses to pathogens with the production of memory cells. BMMSCs modulate the function of dendritic cells (DCs), indirectly regulate T and B cell activity, and delay or prevent the development of acute GVHD (Zhang et al., 2009). BMMSCs have also been shown to suppress the differentiation of DCs and their function during allogeneic islet transplantation (Urban et al., 2008; Aldinucci et al., 2010). BMMSCs strongly inhibited the maturation and functioning of monocytederived DCs by interfering selectively with the generation of immature cells via inhibitory mediator of MSC-derived PGE2 (Lee et al., 2006). PGE2 has been identified as one of the candidates responsible for T cell inhibition by BMMSCs, and may have an immunostimulatory role by facilitating Th1 differentiation and expanding the Th17 T cell population (English et al., 2009; Yao et al., 2009). The expression of PGE2 was shown to be upregulated by IFNγ and TNFα in the BMMSCs for immunomodulatory function (English et al., 2007). BMMSCs can inhibit the cytotoxic effects of antigen-primed cytotoxic T cells by suppressing the proliferation and activity (Zhao et al., 2005) via the inhibition of the nuclear translocation of nuclear factor-kappa B (Matsuda-Hashii et al., 2004). BMMSCs have been shown to alter the NK cell phenotype and suppress proliferation of NK cells via the secretion of TGFβ1 and PGE2, and via cytotoxicity against HLA class I-expressing targets (Aggarwal and Pittenger, 2005; Sotiropoulou et al., 2006; Ryan et al., 2007; Uccelli et al., 2008). BMMSCs have also been shown to inhibit the proliferation of B cells when stimulated with anti-CD40L and IL-4 (Glennie et al., 2005). One report has suggested that allogeneic BMMSCs inhibit the activation, proliferation and IgG secretion of B cells in a BXSB mouse model of human systemic lupus erythematosus (Deng et al., 2005).

Allogeneic BMMSCs are effective in the treatment of murine models of human disease (Zappia et al., 2005; Ding et al., 2009; Fiorina et al., 2009). BMMSCs were shown to be able to secrete regulatory cytokines that affect regulatory T cells, and to modulate the immunological dysregulation observed in antibody producing B cells and cytotoxic NK cells in the NOD mouse (Anderson and Bluestone, 2005). BMMSCs promote the endogenous repair of pancreatic islets and renal glomeruli in a streptozotocin-induced diabetic mouse model (Lee et al., 2006). Co-infusion of BMMSCs and BM cells was shown to inhibit the beta cell-specific T cell proliferation and to restore insulin and glucose levels (Urban et al., 2008). BMMSCs secrete many cytokines and growth factors such as HGF, which shows anti-apoptotic activity in hepatocytes and plays an essential part in the regeneration of the liver (Trim et al., 2000; Matsuda-Hashii et al., 2004). BMMSCs have also been shown to protect against experimental liver fibrosis in CCl4-induced rats (Zhao et al., 2005), and to suppress CD3 T-cell proliferation in collagen-induced arthritis (Schurgers et al., 2010).

In mammals, there are seven sirtuin family members, named Sirt1-7. Sirtuins plays a critical role in the regulation of fundamental biological responses to nutritional and environmental stimuli in each subcellular compartment (Blander and Guarente, 2004; Imai and Guarente, 2010). Sirt1 is a class III protein deacetylase, and Sirt1 activity can be regulated through NAD+. Sirt1 binds to and deacetylates a number of important transcription factors—such as peroxisome proliferator-activated receptor gamma (PPARγ), PPARα, PPAR gamma coactivator 1 alpha (PGC-1α), and the forkhead box, subgroup O (FOXO) family of transcription factors—to drive metabolic responses such as insulin secretion, gluconeogenesis, and fatty acid oxidation (Haigis and Sinclair, 2010). Some reports indicate that Sirt1 promotes osteogenesis and decreases adipogenesis of BMMSCs *in vitro* (Tseng et al., 2011; Peltz et al., 2012; Puri et al., 2012).

Sirt1 deacetylates β-catenin to regulate differentiation of MSCs in MSCs specific Sirt1 knock-out mice (MSC KO) (Simic et al., 2013). Moreover, Sirt1 has been shown to directly downregulate *Sost* gene expression, and promote bone formation in the treatment of osteoporosis (Cohen-Kfir et al., 2011). One report has shown that CD8 T cell differentiation is regulated by basic leucine zipper transcription factor, ATF-like (BATF), which is a member of the AP-1 family, via Sirt1 expression, BATF deficiency inducing high levels of Sirt1 expression in memory CD8 T cells but not in naive CD8 T cells (Kuroda et al., 2011).

#### **IBM-BMT**

We reported that MRL/lpr mice possess abnormal radioresistant stem cells and have provided impressive evidence regarding the origin of ADs in this strain (Ikehara et al., 1989). BMT plus bone graft, which can recruit donor stroma cells, can prevent the recurrence of ADs (Ishida et al., 1994). However, allogeneic BMT + bone grafts failed to treat ADs in MRL/lpr mice, because these mice become more radiosensitive after the onset of lupus nephritis. Moreover, our previous reports showed that stroma cells can be trapped in the liver when BM cells are injected via the portal vein. Thus, directly injecting whole BM cells into the BM, as in IBM-BMT, has been shown to be a powerful strategy for the treatment of ADs in MRL/lpr mice. IBM-BMT, which not only replaces HSCs but also MSCs, has been proven to be the best method for allogeneic BMT: (1) hematopoietic recovery is rapid because the MSCs directly home to the bone cavity, (2) the restoration of T cell functions is complete even in donor-recipient combinations across the MHC barriers, and (3) no graft failure occurs even if the radiation dose is reduced (Kushida et al., 2001). Moreover, IBM-BMT of young marrow cells reversed the reduction of pro-B cells and pre-B cells. The frequency of follicular-B cells in the IBM-BMT group was significantly increased compared to the old group (Hida et al., 2010). We have already used IBM-BMT to successfully treat ADs, osteoporosis, diabetes, Alzheimer's disease, and for the induction of tolerance for organ transplantation (Takada et al., 2006; Guo et al., 2008; Kushida et al., 2009; Li et al., 2009, 2010) (**Table 1**).

BM cells mainly include HSCs and MSCs. MSCs are essential for supporting hematopoiesis in the BM. HSCs can normally proliferate in major histocompatibility complex (MHC)-compatible MSCs even in allogeneic microenvironments. Because the BMCs are directly injected into bone, IBM-BMT circumvents the risk of MSCs being trapped in the lung and liver. And because both MSCs and HSCs are transplanted, hematopoiesis can be rapidly restored. Moreover, IBM-BMT can prevent the risk of graft rejection, even with the use of a mild conditioning regimen (Kushida et al., 2001).

#### **IBM-BMT FOR TREATMENT OF RA**

RA is an AD that results in a chronic, systemic inflammatory disorder that may affect many tissues and organs. RA primarily affects joints, but it also affects other organs such heart, kidney, and blood vessels (Turesson et al., 2003). Its pathophysiology indicates that TNFα drives synovial inflammation and joint destruction. The synovial cells include both fibroblast-like and macrophage-like synoviocytes. Fibroblast-like synoviocytes show abnormal behavior in RA (Scott et al., 2010). About 50% of RA is caused by genetic abnormalities (van der Woude et al., 2009). The classification criteria for RA by the American College of Rheumatology (2010), and the treatment options, are summarized in the work by Scott et al (Scott et al., 2010). Here we talk about stem cell therapy for the treatment of RA in basic experiments and clinical applications.

SKG/Jcl mice are a murine model for RA. BM cells of C57BL/6J mice were transplanted into SKG/Jcl mice using IBM-BMT, and the hematolymphoid cells in the recipient mice were reconstituted by donor-derived cells. There was no evidence of arthritis

in the SKG/Jcl mice at 12 months after transplantation. Moreover, IBM-BMT has been shown to normalize the percentages of Treg (Foxp3+/CD4+) cells, the percentages of receptor activator of NF-kB ligand+ cells on the CD4+ T cells and the serum levels of TNFα, IL-1, and IL-6. One report demonstrated that IBM-BMT is a viable method of immunological manipulation that suppresses the severe joint destruction and bone absorption in SKG/Jcl mice and lends further credence to the use of this methodology in humans with intractable RA (Kushida et al., 2009). Human UC-derived MSCs have been discussed as a possible treatment for RA in the clinical setting. TNFα and IL-6 decreased and CD4+ CD25+ Foxp3+ T cells increased, in active RA patients after UC-derived MSCs were infused, and the UC-derived MSCs survived for 3–6 months, suggesting that treatment with MSCs would benefit RA patients (Wang et al., 2013). Expression of IL-17, IL-6, and TNFα were inhibited when allogeneic UC-derived MSCs were cultured with peripheral blood mononuclear cells (PBMCs) from RA patients, suggesting that MSCs can prevent the expression of these cytokines and that they have therapeutic potential in the treatment of RA (Wang et al., 2012).

#### **MALIGNANT TUMORS TREATED WITH IBM-BMT + THYMUS TRANSPLANTATION (TT)**

Donor lymphocyte infusion (DLI) is a useful method for the treatment of malignant tumors, but it also induces GVHD. However, IBM-BMT has been shown to prevent not only graft failure but also GVHD in animals, even when the radiation dose is reduced (Nakamura et al., 2004). Thus, IBM-BMT plus DLI were used to treat malignant tumors (fibrosarcomas) induced by a tumor cell line (methA). DLI (CD4− spleen cells) can prevent GVHD, but the tumor growth was not suppressed, indicating that CD4+ cells play important roles in graft-versus-tumor (GVT) and GVHD. Our previous results showed that IBM-BMT plus DLI (CD4−lymphocytes) suppressed not only GVHD but also tumor growth (Suzuki et al., 2005). Moreover, the combination of DC, IBM-BMT and DLI showed even better results than the combination of IBM-BMT and DLI in the treatment of solid tumors (Mukaide et al., 2007).

The thymus regulates the production, proliferation and functions of T cells. BMT + TT has been shown to be useful in the treatment of ADs in the MRL/Lpr mouse, because the allogeneic T cells newly-developed by TT are naïve T cells, which show less Fas expression and more resistance to apoptosis than the activated memory T cells with their high Fas expression. We found that the combination of allogeneic IBM-BMT + adult TT from the same donor is effective in mice with solid tumors, as it can induce high thymopoiesis, preserving strong GVT effects without inducing a severe graft-versus-host reaction (GVHR). Meth A sarcoma cells were subcutaneously inoculated into mice, and IBM-BMT + adult TT was then used to treat these mice when the tumor had grown to 5 mm. In tumor-bearing mice, tumor growth was more strongly inhibited by IBM-BMT + adult TT than by IBM-BMT alone. The numbers of CD8+ T cells that infiltrated the tumors, and the number of apoptotic tumor cells, both significantly increased in the mice treated with IBM-BMT + adult TT. IBM-BMT + adult TT prevented tumor development with mild GVHR resulting from the induction of high thymopoiesis and a strong GVT effect in the tumor-bearing mice. The number of CD4+ FoxP3+ cells was lower in the mice treated with IBM-BMT + adult TT than in those treated with IBM-BMT alone. Furthermore, the numbers of CD8+ cells infiltrating the tumor and the levels of IFN-γ were higher in the mice treated with IBM-BMT + adult TT than in those treated with IBM-BMT alone (Miyake et al., 2009). Although T regs have been reported to suppress the GVHR induced by CD4+T cells, they did not reduce the GVT induced by CD8+ T cells (Edinger et al., 2003). Tumors were suppressed to a greater extent as a result of the increased CD4+ and CD8+ T cells and decreased number of Gr-1+/CD11b+ myeloid suppressor cells and Foxp3+/CD4+ T regs, Moreover, the production of CD62L−CD44+ effector memory T cells and IFN-γ were also higher (Zhang et al., 2011).

IBM-BMT seems to be better than co-transplantation of HSCs and cultured MSCs, mainly because the number of functional MSCs may drop after being cultured *in vitro*, and cultured MSCs also are trapped by the liver and lung in the case of IV-BMT. Umbilical cord blood (UCB) can also be used a source of stem cells for transplantation, although the numbers are generally insufficient to allow this to be used as a general source. IBM thus appears to be the best choice for allogeneic transplantation, despite the limited number of stem cells that can be directly transplanted into the bone cavity. In conclusion, IBM-BMT can efficiently transplant both HSCs and MSCs, is useful to treat intractable diseases such as RA and malignant tumors, and in the future may be useful for treating various intractable diseases.

#### **ACKNOWLEDGMENTS**

We would like to thank Mr. Hilary Eastwick-Field and Ms. Keiko Ando for their help in the preparation of the manuscript.

#### **REFERENCES**

malignant tumors: comparison between fetal, newborn, and adult mice. *Stem Cells Dev.* 20, 599–607. doi: 10.1089/scd.2010.0230

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 12 June 2014; accepted: 15 August 2014; published online: 02 September 2014.*

*Citation: Li M, Guo K and Ikehara S (2014) Intractable diseases treated with intrabone marrow-bone marrow transplantation. Front. Cell Dev. Biol. 2:48. doi: 10.3389/ fcell.2014.00048*

*This article was submitted to Stem Cell Research, a section of the journal Frontiers in Cell and Developmental Biology.*

*Copyright © 2014 Li, Guo and Ikehara. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Recruitment of bone marrow-derived cells to periodontal tissue defects

#### *Yasuyuki Kimura1, Motohiro Komaki 2 \*, Kengo Iwasaki 2, Masataka Sata3, Yuichi Izumi 1,4 and Ikuo Morita4,5*

*1 Department of Periodontology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan*

*2 Department of Nanomedicine (DNP), Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan*

*3 Department of Cardiovascular Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan*

*4 Global Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan*

*5 Department of Cellular Physiological Chemistry, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan*

#### *Edited by:*

*Mario Petrini, University of Pisa, Italy*

#### *Reviewed by:*

*Hiroyuki Moriyama, Kinki University, Japan Jyotsna Dhawan, Institute for Stem Cell biology and Regenerative Medicine, India*

#### *\*Correspondence:*

*Motohiro Komaki, Department of Nanomedicine (DNP), Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Room#N607, M&D tower, 1-5-45 Yushima, Bunkyo-ku, 113-8549 Tokyo, Japan e-mail<EMAIL_ADDRESS>
Bone marrow-derived cells (BMCs) are considered to be a major source of mesenchymal stem cells (MSCs) in adults and are known to be effective in periodontal tissue regeneration. However, whether endogenous BMCs are involved in periodontal tissue repair process is uncertain. We therefore created periodontal tissue defects in the buccal alveolar bone of mandibular first molars in bone marrow chimeric mice, and immunohistochemically examined the expression of stromal cell derived factor-1 (SDF-1) and the mobilization of BMCs. We found that SDF-1 expression was increased around the defects at as early as 1 week after injury and that BMCs were mobilized to the defects, while GFP+/CD45+ were rarely observed. Fluorescence-activated cell sorting (FACS) analysis demonstrated that the number of platelet-derived growth factor receptor (pdgfr) α+/Sca-1+ (PαS) cells in the bone marrow decreased after injury. Taken together, these results suggest that BMCs are mobilized to the periodontal tissue defects. Recruitment of BMCs, including a subset of MSCs could be a new target of periodontal treatment.

**Keywords: bone marrow chimeric mice, periodontal defects, mesenchymal stem cells, recruitment, SDF-1**

#### **INTRODUCTION**

Periodontal disease is a bacterially induced chronic inflammatory disease that destroys the tooth-supporting tissue and is one of the main causes of tooth loss. The inflammation and breakdown of tissue can be prevented by conventional periodontal treatment such as scaling and root plaining (tissue debridement). However, the conventional treatment does not regain the tissue that has been lost during the disease process. Recently MSC-like cells have been discovered in periodontal ligament (PDLSCs) (Seo et al., 2004) and extensive studies have been carried out to investigate the potential use of PDLSCs as a therapeutic agent for periodontal regeneration.

Mesenchymal stem cells (MSCs) show multi-differentiation capability and self-renewability *in vitro*. Bone marrow is considered as the one of main source of MSCs. Bone marrow MSCs have attracted attention as donor cells for regenerative therapy, and the efficacy of bone marrow MSCs has been also shown by experiments in the periodontal tissue regeneration. For example, it has been reported that canine periodontal defects can be regenerated from bone marrow MSCs mixed with atelocollagen (Kawaguchi et al., 2004) and that transplanted bone marrow MSCs were observed in regenerated periodontal tissue (Hasegawa et al., 2006). Yang et al. demonstrated that engraftment of bone marrow-derived MSCs with gelatin beads successfully regenerated periodontal tissue in rats (Yang et al., 2010).

Endogenous bone marrow-derived cells (BMCs) including MSCs have been reported to promote repair of the remote tissue by mobilizing into peripheral blood by injury signals, and homing to injured tissues (Mansilla et al., 2006; Alm et al., 2010). Recently, SDF-1 has been reported to involve in the recruitment and engraftment of stem cells in wound sites (Yin et al., 2010). C-X-C chemokine receptor type 4 (CXCR4) is a unique receptor of SDF-1, and is known to express in various cell types, including stem cells (Honczarenko et al., 2006). However, whether the SDF-1/CXCR4 axis is involved in recruitment of BMCs to periodontal wound is not clarified. Particularly, the contribution of endogenous bone marrow MSCs during periodontal tissue repair is not fully understood due to lack of appropriate detection system of MSCs *in vivo*.

A lack of the unique marker is a major obstacle for detection of MSCs *in vivo*. The characteristics of MSCs were confirmed by combination expression of cell surface markers such as CD44, CD73, CD90, and CD105 in a single cell. Besides it must be shown that there is not expression of hematopoietic stem cell marker, CD34 and hematopoietic progeny markers such as CD11b, and CD45. Recently, Morikawa et al. reported the method to isolate MSCs from murine bone marrow without cell culture by cell sorting of pdgfrα (+)/Sca-1 (+) cells (Morikawa et al., 2009).

In this study, we created periodontal defects in the buccal alveolar bone of mandibular first molars in bone marrow chimeric mice and investigated the expression of SDF-1 and the recruitment of bone marrow MSCs during periodontal tissue repair process.

#### **MATERIALS AND METHODS**

#### **PREPARATION OF BONE MARROW CHIMERIC MICE**

BMCs were isolated from femurs and tibias of GFP transgenic mice as reported previously (Okabe et al., 1997; Sata et al., 2002; Fukuda et al., 2009). In brief, BMCs were hemolyzed with ACK lysing buffer (Lonza, Basel, Switzerland). C57/BL/6 mice (age, 8 weeks; male) were lethally irradiated 9.5 Gy (MBR-1520RB; Hitachi, Tokyo, Japan). Two days after irradiation, unfractionated BMCs (1 × 106 cells/0.3 ml D-PBS) from GFP transgenic mice were intravenously injected into irradiated mice by tail vein puncture. Eight weeks after the transplantation, peripheral bloods were collected from retro-orbital plexus. Replacement ratio of bone marrow was confirmed by fluorescence-activated cell sorting (FACS) aria (BD, Franklin Lakes, NJ, USA). Chimeric mice with a bone marrow substitution rate of over 83% were used in this experiment. All procedures involving the experimental animals were performed in accordance with protocols approved by the local institutional guidelines for animal care of The University of Tokyo and Tokyo Medical and Dental University (0120218A) and complied with the *Guide for the Care and Use of Laboratory Animals* (NIH guidelines 32).

#### **EXPERIMENTAL PERIODONTAL TISSUE INJURY**

Under general anesthesia with sodium pentobarbital (40– 50 mg/kg, IP), we produced 2*.*0 × 1*.*5 mm periodontal tissue defects in the buccal alveolar bone of mandibular first molars in bone marrow chimeric mice, by removing alveolar bone, periodontal ligament and cementum using a round bar with water cooling under a stereoscopic microscope.

#### **IMMUNOHISTOCHEMICAL STAINING**

Before or at 1, 2, 4, 5, and 10 weeks after injury, tissue was fixed with 4% paraformaldehyde, followed by decalcification in 10% ethylenediaminetetraacetic acid (EDTA) solution at 4◦C. Tissue sections (5μ) were immunostained for GFP and SDF-1. Briefly, after deparaffinization, sections were washed with PBS, and were treated with 1% hydrogen peroxide (Wako, Osaka, Japan) in methanol. After washing with PBS, sections were blocked with blocking solution (0.5% goat serum in PBS) at room temperature for 30 min.

Before anti mouse SDF-1 antibody treatment, sections were treated with a M.O.M. Immunodetection Kit (Vector Laboratories Inc., Burlingame, CA, USA). Sections were then treated with either anti-mouse GFP antibody (1: 500 dilution, #47894A; Molecular Probes, Eugene, OR) or anti mouse SDF-1 antibody (1: 1000 dilution, #79014; R&D systems, Minneapolis, MN, USA) or anti-mouse CD45 antibody (1: 200 dilution #550539; BD) for 2 h. After washing with PBS three times, sections were treated with secondary antibodies, either anti-rabbit conjugated with biotin (Dako Japan, Kyoto, Japan) or anti-mouse conjugated with biotin (Dako Japan) or anti-rat Cy3 (1: 200 diluted, #56021; Jackson, West Grove, PA, USA) at room temperature for an hour. After washing with PBS three times, sections were treated with ABC-AP mix (Vector Laboratories Inc.) at room temperature for an hour. Detection was performed using Vector Red at room temperature for 10–20 min. Histological examination was performed

#### **FLUORESCENCE-ACTIVATED CELL SORTER (FACS) ANALYSIS FOR IDENTIFICATION OF BONE MARROW MSCs**

In the experimental group, BMCs were obtained from murine femurs and tibias 1 week after preparation of periodontal defects. Untreated mice were used as controls. To evaluate the ratio of bone marrow MSCs for all collected cell counts, we conducted at least three experiments using two mice in the experimental and control groups. BMCs were obtained as described previously (Morikawa et al., 2009). Briefly, femurs and tibias were aseptically removed from two mice and bones were crushed with an icecold pestle and mortar. Bone chips, including marrow were rinsed with HBSS+ and were digested using collagenase (#032-22364; Wako) for an hour at 37◦C. Collected BMCs were hemolyzed and FcR was blocked with anti-mouse CD16/32 (#553142; BD) on ice for 5 min. BMCs (2–5 × 105) were multi-stained with CD45.2- APC-eFlour780 (#47-0454; eBioscience, San Diego, CA, USA), TER119-PECy7 (#25-5921; eBioscience), pdgfrα-PE (#12-1401- 81; eBioscience), Sca-1-APC (#17-5981-81; eBioscience) (1: 100 dilution, 30 min, on ice) and 7AAD (#559925; BD) (1: 100 dilution, 10 min, on ice). The ratio of MSCs [CD45.2 (−), TER119 (−), 7AAD (−), pdgfrα (+), Sca-1 (+)] in BMCs was evaluated by FACS analysis.

#### **RESULTS**

#### **LOCALIZATION OF GFP-POSITIVE CELLS IN PERIODONTAL TISSUE**

In order to investigate the localization of BMCs, we prepared bone marrow chimeric mice and examined GFP-positive cells in the periodontium. Both osteoclasts-like multinucleated giant cell in resorption pits of alveolar bone and macrophages in gingival epithelium, which are known to be derived from bone marrow, were positive for GFP (**Figures 1A,B**). In control mice, GFPpositive cells were observed in both periodontal ligament and dental pulp (**Figures 1C–E**). GFP-positive cells in periodontal ligament were mainly observed around blood vessels (**Figure 1D**). GFP-positive cells were rarely observed in alveolar bone or dentin.

#### **TIME-COURSE OBSERVATIONS OF BMCs AROUND PERIODONTAL TISSUE DEFECTS**

Experimental periodontal tissue defects were created in bone marrow chimeric mice. Five weeks after injury, GFP-positive cells were observed in the periodontal ligament in both control and experimental groups. In the experimental group, the number of GFP-positive cells increased significantly around the periodontal tissue defects (**Figure 2**). Ten weeks after injury, GFP-positive cells around the periodontal tissue defects decreased to control levels. Meanwhile, no changes were seen during the course of experiment in control tissue. In addition, we performed double staining for SDF-1 and GFP in order to check co-localization of SDF-1 and GFP. Five weeks after injury, SDF-1 expression was ubiquitously seen in periodontal ligament and the number of BMCs in experimental tissues was higher than in controls (**Figure 3**). In control tissue, SDF-1 expression was dominantly observed around blood vessels. A double staining for GFP and CD45 showed limited co-localization of GFP (green) and CD45

**FIGURE 1 | Localization of GFP-positive cells in intact periodontal tissue.** Visualization of BMCs *in vivo* on periodontal tissue. Arrows indicate osteoclast-like multinucleated giant cells **(A)** and macrophage-like cells in gingival epithelium **(B)** were GFP-positive, implying that GFP-positive images were confirmed to match BMCs. GFP-positive cells were also observed at

the periodontal ligament **(C)**, blood vessels **(D)**, and dental pulp **(E)**. A, multinucleated giant cells in resorption lacunae of alveolar bone; B, gingival epithelium; C, periodontal ligament; D, blood vessels in periodontal ligament; E, dental pulp. G, gingiva; AB, alveolar bone; P, periodontal ligament; DP, dental pulp; D, dentin.

(red). In experimental tissue the number of GFP(+)/CD45(+) cells peaked at 13% GFP-positive cells.

#### **TIME-COURSE OBSERVATIONS OF SDF-1 EXPRESSION AROUND PERIODONTAL TISSUE DEFECTS**

At 1, 2, and 4 weeks after injury, SDF-1 expression was immunohistochemically evaluated (**Figure 4**). One week after

**FIGURE 3 | Double staining of GFP and SDF-1/CD45 in periodontal ligaments.** In control sites, blood vessel-like structures showed intense staining of SDF-1. GFP-positive cells were observed around SDF-1-positive cells. In experimental sites, SDF-1 expression was ubiquitously detected in periodontal ligament 5 weeks after surgery. The number of GFP-positive cells in experimental tissues was higher than in controls. In experimental tissue, the number of GFP(+)/CD45(+) cells (yellow arrows) was elevated, accounting for 13% of GFP-positive cells (*n* = 4).

injury, some SDF-1 was observed within the blood vessels of periodontal ligaments in control tissue. In experimental tissue, weak and diffuse SDF-1 staining was observed in the defects and periodontal ligament. After 2 weeks, SDF-1 staining increased markedly and was attenuated by 4 weeks after injury.

#### **POST-OPERATIVE CHANGES IN MSC POPULATION IN BONE MARROW**

One week after injury, BMCs were collected from murine femurs and tibias. Immediately after collecting BMCs, MSC population in bone marrow was evaluated by FACS analysis. FACS analysis confirmed that the percentage of pdgfrα (+)/Sca-1(+) cells in bone marrow of mice with periodontal defects was significantly lower when compared to controls (**Figure 5**).

#### **DISCUSSION**

Involvement of BMCs in the physiology and pathology of various tissues, including heart, lung, liver, kidney, skeletal muscle, bone, and blood vessels, has been reported. Some of these studies used bone marrow chimeric mice and successfully tracked BMCs. In order to determine roles of endogenous BMCs in periodontal tissue, we created bone marrow chimeric mice. Both osteoclast-like multinucleated giant cells at the alveolar bone and macrophages in gingival connective tissue, known to be of bone marrow origin, were positive for GFP staining (**Figures 1D,E**), implying BMCs were successfully tracked. However, we should be aware of possibility that recipient bone marrow cells may fuse with donor cells, acquiring such phenotypes in periodontal tissue. GFP-positive cells were observed in both the periodontal ligament and dental pulp (**Figures 1A,B**). To our knowledge, this is the first time that the presence of endogenous BMCs has been demonstrated in naive murine periodontal ligament, at least by observation of bone marrow chimeric mice. In addition, these cells were mainly observed around blood vessels in periodontal ligament (**Figure 1C**). McCulloch reported that stem cell-like cells with a slow rate of cell proliferation were located in paravascular

sites in murine periodontal ligament (McCulloch, 1985). Chen et al. reported that putative stem cells that showed cross-reactivity with at least one among STRO-1, CD146, and CD44 antibodies, mainly in the paravascular region of human periodontal ligament (Chen et al., 2006). It remains elusive whether BMCs observed in our study were the same as the MSCs/progenitor cells residing in periodontal tissue. It is of interest to clarify the physiological role of BMCs in periodontium and to determine whether BMCs are the origin of MSCs/progenitor cells around blood vessels residing in the periodontal ligament. Further studies are thus necessary.

Next, in order to determine whether BMCs contribute to periodontal wound healing, we created periodontal defects in the bone marrow chimeric mice. Zhou et al. have reported engraftment and differentiation of BMCs to periodontal tissue-forming cells when periodontal defects were treated by regenerative procedure, a grafting of ceramic bovine bone (Zhou et al., 2011). In contrast, Ohta et al. have reported that bone marrow-derived MSCs were not detected in repairing periodontal ligament by a single staining of either STRO-1 or CD44 (Ohta et al., 2008). Our result clearly demonstrated that the number of BMCs around periodontal defects increased as a result of tissue injury in accordance with data reported by Zhou et al. The result that Zhou et al. demonstrated is different from ours in that they investigated the participation of BMCs after periodontal regenerative treatment. In contrast, we investigated whether BMCs are involved in tissue repair without treatment. We observed neither localization of BMCs in alveolar bone 10 weeks post-surgery nor restoration of alveolar bone. It is well known that the tissue debridement abrogates tissue degradation, but does not regain the lost tissue, implying that our model mimics this clinical situation. It is also possible that periodontal tissue healing in irradiated recipient mice may not represent a physiological process that occurs naturally in non-irradiated mice in response to injury. Thus, it remains to be elucidated whether mobilized BMCs actually contribute to periodontal tissue healing.

Kitaori et al. reported that SDF-1 increased during the repair of bone grafts at both the messenger RNA and protein levels, and that anti-SDF-1 antibody inhibited new bone formation (Kitaori et al., 2009). Moreover, Jones et al. demonstrated that migration of MSCs to bone and bone marrow is CXCR4/SDF-1 dependent in a murine osteogenesis imperfecta model and that SDF-1 up-regulates CXCR4 demonstrating chemotaxis *in vitro* and enhancing engraftment *in vivo* (Jones et al., 2012). It has been reported that inflammation and hypoxia play an important role in regulating the SDF-1/CXCR4 axis. First we confirmed that hypoxia around periodontal defects after periodontal injury (supplemental figure 1). We then checked the expression of SDF-1 in periodontium, and found that SDF-1 expression increased around periodontal defects and in periodontal ligament prior to the recruitment of BMCs (**Figure 4**). It has been reported that SDF-1 levels increase in periodontal disease. (Havens et al., 2008) We confirmed that SDF-1 stimulated the migration of plastic dish-adhered BMCs and that CXCR4-positive BMCs migrated to SDF-1 more promptly than CXCR4-negative BMCs in transwell assay (supplemental figure 2). It has been reported that in BMCs hematopoietic cells, hematopoietic stem cells, MSCs, and endothelial progenitor cells express CXCR4 and that other molecules such as inflammatory factors and cytokines are involved in mobilization of BMCs. (Salem and Thiemermann, 2010) Further experiments are necessary to examine the direct involvement of the SDF-1/CXCR4 axis in recruitment of BMCs *in vivo*. It is also interesting to compare recruitment potential among BMCs.

Adult bone marrow contains several distinct populations of stem cells, such as hematopoietic stem cells, MSCs, endothelial progenitor cells. We observed the number of GFP-positive cells increased in periodontal ligament 5 weeks after injury and about 13% of them are CD45 positive (**Figure 3**). This result suggested that most of cells detected in periodontal ligament were not hematopoietic cells at least at time point we evaluated (5 weeks after injury). It is necessary to directly determine what types of cells are recruited to periodontal defects.

In FACS analyses, we used CD45 (−), TER119 (−), PDGFRα (+), and Sca-1 (+) cells to detect PαS cells, a subset of MSCs in bone marrow. Based on the results of FACS analyses, we demonstrated for the first time that PαS cells in bone marrow decreased after periodontal injury (**Figure 5**). However, it was not clarified whether PαS cells contribute to periodontal tissue repair process. Morikawa et al. reported that PaS-derived clones with multi-differentiation potential are also positive for CD105 and CD90 *in vitro* (Morikawa et al., 2009). It is very important to analyze whether PαS cells that migrated to periodontal defects also express CD90 and CD105.

In summary, our data demonstrate that bone marrow cells are mobilized to periodontal defects. Further experiments are needed to determine cells to be mobilized from bone marrow to periodontal defects and regulatory mechanism involved in this process. Recruitment of BMCs, including a subset of MSCs could be a new target of periodontal treatment.

#### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http://www*.*frontiersin*.*org/journal/10*.*3389/fcell*.*2014*.*00019/ abstract

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 18 December 2013; accepted: 28 April 2014; published online: 21 May 2014. Citation: Kimura Y, Komaki M, Iwasaki K, Sata M, Izumi Y and Morita I (2014) Recruitment of bone marrow-derived cells to periodontal tissue defects. Front. Cell Dev. Biol. 2:19. doi: 10.3389/fcell.2014.00019*

*This article was submitted to Stem Cell Research, a section of the journal Frontiers in Cell and Developmental Biology.*

*Copyright © 2014 Kimura, Komaki, Iwasaki, Sata, Izumi and Morita. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Cancer-associated mesenchymal stem cells aggravate tumor progression

Chie Kudo-Saito\*

*Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan*

Mesenchymal stem cells (MSCs) have both stemness and multi-modulatory activities on other cells, and the immunosuppressive and tumor-promotive mechanisms have been intensively investigated in cancer. The role of MSCs appears to be revealed in tumor aggravation, and targeting MSCs seems to be a promising strategy for treating cancer patients. However, it is still impractical in clinical therapy, since the precise MSCs are poorly understood in the *in vivo* setting. In previous studies, MSCs were obtained from different sources, and were prepared by *ex vivo* expansion for a long term. The inconsistent experimental conditions made the *in vivo* MSCs obscure. To define the MSCs in the host is a priority issue for targeting MSCs in cancer therapy. We recently identified a unique subpopulation of MSCs increasing in mice and human with cancer metastasis. These MSCs are specifically expanded by metastatic tumor cells, and promote tumor progression and dissemination accompanied by immune suppression and dysfunction in the host, more powerfully than normal MSCs growing without interference of cancer. In this review, we summarize current knowledge of the role of MSCs in tumor aggravation, along with our new findings of the bizarre MSCs.

Keywords: mesenchymal stem cell, cancer, metastasis, epithelial-to-mesenchymal transition, immunosuppression

# Introduction

Mesenchymal stem cells (MSCs) have stemness including self-renewability and pluripotency to differentiate into adipocytes, osteocytes, chondrocytes, fibroblasts, pericytes and more, and also have multiple immunoregulatory properties for maintaining immune tolerance (Uccelli et al., 2008; Liu et al., 2009). MSCs suppress immune responses directly by producing immunomodulatory molecules such as IDO, PGE2, TGFβ and nitric oxide, and indirectly via generation and expansion of potent immunosuppressors such as CD4+FOXP3+ or CD8+FOXP3+ regulatory T cells (Tregs) and myeloid-derived regulatory cells including dendritic cells (DCregs), monocytes/macrophages (M-MDSCs) and granulocytes (G-MDSCs) (Nauta and Fibbe, 2007; Uccelli et al., 2008; Maggini et al., 2010). Recent studies have demonstrated that MSCs are originally silent, and become immunosuppressive by activation with pro-inflammatory cytokines such as IFNγ, TNFα, and IL-1β, and ligation of TLRs such as TLR2, TLR3, and TLR4 expressed on the MSCs (Burr et al., 2013; English, 2013). This suggests that the functional role of MSCs depends on the components within the microenvironment.

Tumor tissues contain a number of influential factors for recruiting and activating MSCs (Uccelli et al., 2008; Yang et al., 2013). In turn, in the tumor microenvironment, the MSCs modulate

#### Edited by:

*Simone Pacini, University of Pisa, Italy*

#### Reviewed by:

*Eumorphia Remboutsika, Biomedical Sciences Research Center "Alexander Fleming," Greece Laura Iop, University of Padua, Italy*

#### \*Correspondence:

*Chie Kudo-Saito, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Stem Cell Research, a section of the journal Frontiers in Cell and Developmental Biology*

> Received: *12 February 2015* Paper pending published: *02 March 2015* Accepted: *17 March 2015* Published: *01 April 2015*

#### Citation:

*Kudo-Saito C (2015) Cancer-associated mesenchymal stem cells aggravate tumor progression. Front. Cell Dev. Biol. 3:23. doi: 10.3389/fcell.2015.00023* biological properties of tumor cells directly by causing epithelialto-mesenchymal transition (EMT) followed by induction of tumor metastasis and cancer-initiating stem cells (CSCs), and indirectly via angiogenesis followed by promotion of tumor growth (Uccelli et al., 2008; Yang et al., 2013). These totally lead to tumor heterogeneity responsible for resistance to various treatments. Thus, targeting immunoregulatory and tumor-promotive MSCs seems to be a promising strategy for both attenuating the tumor malignancy, and improving the host immunity against cancer in treating patients.

However, targeting MSCs is still unrealistic in clinical settings, because the in vivo MSC profiles remain obscure. The surface molecules expressed in MSCs have been widely investigated, and many studies demonstrated the high expression of CD49a, CD73, CD90, CD105, CD146, CD271, and STRO-1, but not CD11b, CD14, CD19, CD34, CD45, CD79a in human MSCs (Nauta and Fibbe, 2007; Kuhn and Tuan, 2010). However, these molecules are not unique markers specific for MSC phenotype and function. In previous studies, MSCs were mostly obtained from different sources followed by ex vivo expansion for a long term. This implies that only a part of MSC subpopulations might be selectively expanded, or transformation in MSCs might generate another type different from the original MSCs in the host. Furthermore, the biological properties of MSCs may be much more modulated under cancer than we have ever known in regenerative research without cancer. These inconsistent experimental conditions impede development of targeting MSCs in cancer therapy. To rigorously define the precise MSCs in cancer patients is a priority issue for the practical application of MSC targeting in the treatment.

We have been exploring novel anticancer therapeutics by focusing on the interplay between neoplastic lesions and host immunity for a while, and have recently provided new insights into the metastatic process (Kudo-Saito, 2013; Kudo-Saito et al., 2013a, 2014). Using murine and human tumor cells with typical features of EMT, high motility and invasivity, following transduction of a cDNA coding for snail family zinc finger 1 (Snail), we found that Snail+ metastatic tumor cells specifically release a large amount of TSP1 (Kudo-Saito et al., 2009), CCL2 (Kudo-Saito et al., 2013b), and FSTL1 (Kudo-Saito, 2013; Kudo-Saito et al., 2013a), all of which can generate immune suppression and dysfunction mediated by immunoregulatory cells including CD4+Foxp3+ Tregs, CD11c+MHC IIlow/− DCregs and CD45−ALCAM+ MSCs, and functionally impaired CD8low T cells. This pathway totally accelerates cancer metastasis in the host. Among the Snail+ tumor-producing factors, FSTL1, which is a member of the SPARC family (Sylva et al., 2013), is an outstanding molecule playing a dual role in cancer metastasis particularly to the bones. FSTL1 confers the invasive potential and bone tropism on tumor cells, and also generates and expands CD45−ALCAM+ MSCs initially in bone marrow and sequentially all over the body in the host (Kudo-Saito, 2013; Kudo-Saito et al., 2013a). We compared the biological properties between Snail/FSTL1-expanded MSCs (designated "sMSCs") and other MSCs manipulated by none or non-metastatic tumor cells prepared in vitro and in vivo. The in vitro MSCs were CD45− cells sorted from bone marrow, spleen and peripheral blood of naive mice followed by stimulation with none or culture supernatant of Snail− or Snail+ tumor cells for 5–7 days. The in vivo MSCs were CD45− cells freshly isolated from those tissues of naive mice or the mice implanted with Snail− or Snail+ tumor cells (labeled with GFP for elimination). We found that the sMSCs prepared either in vitro or in vivo specifically produce ANGPT2, CCL2, CCL3, and FSTL1, and most powerfully affect tumor behavior and host immunity by using these molecules (unpublished data except CCL2). ALCAM is expressed only in these MSCs, but not in normal MSCs growing without interference of cancer. These MSCs could be the "activated MSCs" with immunoregulatory properties, since ALCAM is known as an activation marker as well as a MSC marker (Weidle et al., 2010). We validated these results observed in the murine system, using human system with human tumor cells and PBMCs. In addition, we immunohistochemically analyzed tumor tissues of advanced breast cancer patients, and found that accumulation of ALCAM+ cells (possibly including the sMSCs) significantly correlates with FSTL1 expression level in tumor portions, but not in the adjacent normal counterparts. This points to the in vivo existence of a causal molecular connection between FSTL1 and ALCAM within the tumor microenvironment in patients.

In another study focusing on human endogenous retrovirus antigen H (HERV-H) that is frequently and highly expressed in metastatic tumor cells, we also found that CD45−CD271+ MSCs are specifically recruited by CCL19 released from HERV-H+ tumor cells in the microenvironment (Kudo-Saito et al., 2014). These MSCs are expanded by a 17-mer peptide that is encoded in the immunosuppressive domain of the HERV-H envelope protein. HERV-H was also found to upstreamly regulate Snail and Twist expressions in the metastatic tumor cells. In the immunohistochemical analysis, the significant correlation between HERV-H and CCL19 expressions and accumulation of CD271+ cells (possibly including the HERV-H-induced MSCs) was observed in tumor tissues of colon cancer patients. This also suggests the clinical relevance of the experimental findings. Later, we additionally found a high similarity of the phenotype and function between the sMSCs and the HERV-H-induced MSCs. Both ALCAM and CD271 are the functional molecules required for cell proliferation and sphere formation of these MSCs, and the soluble factors released by the sMSCs are also upregulated in the HERV-H-induced MSCs (unpublished data). This suggests that the CD45−ALCAM+CD271+ MSCs (also designated "sMSCs" below) could be the cancer-activated MSCs in vivo, particularly generated and expanded in the presence of cancer metastasis in patients. The sMSCs would be a promising target for breaking through the difficulty in treating patients with cancer metastasis and impaired immunity. Here, we summarize the current knowledge of the diversified functional role of the bizarre sMSCs (**Figure 1**) including normal MSCs in tumor aggravation.

# MSCs Promote Tumor Growth

Because of the high migratory property, MSCs promptly migrate into tumor tissues in response to the inflammatory molecules including chemokines such as CCL5 (Karnoub et al., 2007; Luo et al., 2014) and CXCL16 (Jung et al., 2013). MSCs were

considered to contribute to tumor progression by causing angiogenesis essential for tumor survival and growth after differentiation into the cancer-associated cells such as fibroblasts (CAFs), pericytes and endothelial cells (Bergfeld and Declerck, 2010). However, the role of the MSC itself begins to be recognized in tumor progression. One paper has demonstrated that CD44 expressed in MSCs is a functional molecule crucial for the migratory and angiogenic properties of the differentiated CAFs (Spaeth et al., 2013). This suggests that the functional role of fibroblasts depend on the original MSCs. In our study, a number of angiogenic factors were significantly upregulated in the sMSCs, and also in the MSCs manipulated by non-metastatic tumor cells as compared to normal MSCs. This seems to be the cancer-associated features. Particularly, ANGPT2 and CCL2 were specifically released by the sMSCs leading to the much more aggressive tumor growth, although no superior production of other potent angiogenic molecules like VEGFs was seen in the sMSCs compared to other MSCs (unpublished data). When the sMSCs were transfected with siRNAs specific for angpt2 or ccl2 before coinjection with tumor cells in mice, the sMSC-induced tumor growth accompanied by increase of F4/80+TIE2+ tumorassociated macrophages (TAMs) or CD11b+Gr1+ MDSCs in the tumor tissues was significantly inhibited. This suggests that the sMSC-derived ANGPT2 and CCL2 play a key role in the sMSCinduced tumor growth. ANGPT2, which binds to TEK/TIE2, is the antagonist of a potent angiogenic molecule ANGPT1 (Fagiani and Christofori, 2013). However, as well as CCL2, ANGPT2 has been demonstrated to promote tumor growth indirectly via increase of TIE2+ TAM-like macrophages in the tumor microenvironment (De Palma and Naldini, 2011). FSTL1 was reported to significantly increase in CAFs within stroma of colon cancer tissues (Torres et al., 2013). However, FSTL1 was a small impact on tumor growth in our study.

Some studies have reported no MSC efficacy on tumor growth (Bergfeld and Declerck, 2010). One possible reason for the paradoxical effects may be the experimental conditions such as MSC sources and in vitro culture term. Another possible reason may be the distribution site and the amount of the MSCs in vivo. In our experiments, the sMSC effect on tumor growth depended on the route of injection of MSCs into mice. Subcutaneous tumor growth was much more aggressively promoted by subcutaneous (s.c.) coinjection with the sMSCs as compared to the case of intravenous (i.v.) injection, by which only a small and limited amount of the sMSCs spontaneously migrated into the tumor tissues (Kudo-Saito et al., 2013a).

## MSCs Promote Tumor Metastasis

Recent studies begin to uncover the roles of MSCs in facilitating tumor metastasis. CCL5 is one of the chemokines produced from both tumor cells and MSCs in the tumor tissues of breast cancer (Karnoub et al., 2007) and prostate cancer (Luo et al., 2014). Tumor-derived CCL5 recruits MSCs to the microenvironment, and the MSCs also release CCL5 for inducing EMT in the tumor cells. Possibly because the major source of MSCs is bone marrow, the relationship between MSCs and bone metastasis has been intensively investigated (Bergfeld and Declerck, 2010). Bone metastasis is frequently seen in patients, particularly with breast cancers and prostate cancers, giving a high risk that deteriorates the quality of life of patients leading to poor prognosis (Weilbaecher et al., 2011). Previous studies demonstrated that tumor cells migrate into bone marrow in response to various chemokines and cytokines such as CXCL12, CCL2, and IL6, which are released by MSCs (Bergfeld and Declerck, 2010). However, little is known about the molecular mechanisms how MSCs direct tumor cells to the bone.

In our study, we did not observed the significant production of CCL5 and CXCL12 in the sMSCs. Instead, ANGPT2, CCL2, and FSTL1 are significantly upregulated in the sMSCs compared to other MSCs (Kudo-Saito et al., 2013a). The crucial roles of ANGPT2 (Minami et al., 2013; Rigamonti and De Palma, 2013) and CCL2 (Lu et al., 2009; Tang and Tsai, 2012) have been already established in the mechanism of cancer metastasis. We also demonstrated the significance of CCL2 in EMTassociated cancer metastasis (Kudo-Saito et al., 2013b). Later, we additionally found that FSTL1 plays a key role in bone metastasis. FSTL1 can confer bone polarity on tumor cells by inducing the bone metastasis-associated molecular expression such as CCR2, CXCR4, and RANKL (Kudo-Saito et al., 2013a). Cancer metastasis is amplified by CCL2 and FSTL1, which are abundantly produced by both the sMSCs and the metastatic tumor cells within the tumor microenvironment. Interestingly, in addition to the soluble factors, cell-cell contact between tumor cells and the sMSCs is required for tumor metastasis, particularly to bone marrow, where is a niche for maintaining CSCs (Kudo-Saito et al., 2013a). This is also involved in the mechanism of the sMSC-created tumor heterogeneity as described below.

# MSCs Create Tumor Heterogeneity and Complexity

It is increasingly being recognized that every tumor cell has a different profile in gene expression and cellular function even if the source (tumor tissue and patient) is just same, and the clonal evolution in a tumor cell builds tumor heterogeneity and complexity governing resistance to the treatments leading to poor outcome in patients (Junttila and De Sauvage, 2013). Although the extracellular matrix is one of the most influential factors for altering tumor properties (Faurobert et al., 2015), MSCs have been also demonstrated to play a key role in generating CSCs with a high metastaticity, dormancy and chemoresistance. In our study, we also observed that tumor cells transform into CSC-like tumor cells following in vitro coculture or in vivo coinjection with the sMSCs (Kudo-Saito et al., 2013a). This is partly mediated by cellcell interaction through the ALCAM-ALCAM homophilic binding after ALCAM clustering on the cell surface, which is crucial for proliferation and colonization of CSCs (Weidle et al., 2010) and the sMSCs (Kudo-Saito et al., 2013a). Interestingly, Roodhart et al. reported platinum-based chemotherapy activates MSCs to produce polyunsaturated fatty acids, which confer chemoresistance on tumor cells (Roodhart et al., 2011). This gives a warning in practical settings of treating cancer patients with chemotherapeutics.

In addition to such epigenetic mutations in tumor cells, spontaneous hybridization between tumor cells and MSCs has been recently demonstrated as a non-mutational mechanism in breast cancer (Rappa et al., 2012) and lung cancer (Xu et al., 2014). The tumor/MSC-fused cells have CSC-like properties such as high tumorigenicity and metastaticity, maintaining both profiles of tumor cells and MSCs. In our study, when tumor cells were cocultured with the sMSCs in phagocytosis assay, a few tumor cells were engulfed by the sMSCs, but not fused together, and the tumor cells taken were kept inside of the sMSCs alive without cell death or digestion for a long term (unpublished data). This may be another mechanism that tumor cells lurk with the help of MSCs in the host. To analyze the phenotype and behavior of these tumor-holding MSCs may contribute to further understanding of the mechanism involved in tumor survival and escape. Interestingly, it has been also demonstrated that MSCs transform into tumor-like cells with high proliferative ability following spontaneous mutations (Miura et al., 2006; Tolar et al., 2007; Rosland et al., 2009; Mohseny and Hogendoorn, 2011). This seems to be the fundamental mechanism underlying the emergence of the cancer stem cells originated from non-cancerous cells in the host.

# MSCs Protect Tumor Cells from Immune Attack

MSCs cause immune suppression and dysfunction, which totally support all steps of tumor progression (Nauta and Fibbe, 2007; Uccelli et al., 2008). Such immunoregulatory MSCs have been interest in the therapy to properly induce and activate anti-tumor immune responses in cancer patients, since impaired immunity has been a critical issue in the treatment (Uccelli et al., 2008; Yang et al., 2013). Recent studies have demonstrated that MSCs become immunosuppressive following activation through inflammatory signals (Burr et al., 2013; English, 2013). A tumor tissue, where is a milieu filled with abundant pro-inflammatory molecules produced by tumor cells and a variety of the infiltrating cells, could be the best educational place for MSCs to acquire the distinctive competence to support tumor cells. However, how to distinguish between the cancer-activated MSCs and normal healthy MSCs remains to be elucidated.

In our study, we identified CCL2, CCL3, and FSTL1 as the prominent immunomodulatory molecules upregulated only in the sMSCs. The sMSCs generate functionally impaired CD8low T cells through cell-cell contact with ALCAM expressed on the sMSCs (Kudo-Saito et al., 2013a), in addition to regular Tregs as reported elsewhere (Nauta and Fibbe, 2007; Uccelli et al., 2008; Maggini et al., 2010). In the previous studies showing Treg or CD8low T-cell induction by MSCs, CD8 reduction in the Foxp3+ T cells (Prevosto et al., 2007), or Foxp3 expression in the CD8low T cells (Giuliani et al., 2011) were not analyzed. The sMSC-derived FSTL1 might amplify such abnormal immunity by promoting the sMSC expansion in an autocrine manner as well as a paracrine manner by the metastatic tumorderived FSTL1. CCL2 is also responsible for immunosuppression. In our study, CCL2 orchestrates CCR2-expressing immunomodulatory cells including Tregs, MDSCs and TAMs in the tumor microenvironment, and generates DCregs partly in collaboration with LCN2 that is released from the CCL2-stimulated tumor cells (Kudo-Saito et al., 2013b). Tregs within tumor microenvironment have been demonstrated to highly express a CCL3 receptor, CCR5 (Tan et al., 2009; Schlecker et al., 2012). The sMSC-derived CCL3 may also help to promptly create a beneficial environment for immunoevasion of tumor cells by recruiting the cancerassociated CCR5+ Tregs, although not yet investigated in our study.

#### Concluding Remarks

The significant advances in profiling MSCs provided abundant knowledge about MSCs, and revealed the central role of

#### References

MSCs in tumor development and progression. The emerging evidences show that MSCs stand out as a promising therapeutic target for treating cancer and the consequent impaired immunity in patients. Nevertheless, we have understood little about the MSCs interacting with tumor cells in the in vivo setting. Our sMSC study is gradually unmasking the characteristics of the cancer-activated MSCs that can sharply build heterogeneic and complexed microenvironment beneficial for tumor survival and escape in the host. However, further studies are still needed to achieve translation of the findings to the clinical therapy.

#### Acknowledgments

The sMSC research was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (19590405, 21590445, and 26430122), The Sagawa Foundation for Promotion of Cancer Research, and Keio Gijuku Academic Development Funds.

EMT of cancer cells. Cancer Cell 15, 195–206. doi: 10.1016/j.ccr.2009. 01.023

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Kudo-Saito. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.