MESENCHYMAL STEM CELLS
SCIENTIFIC REVIEW

INTRODUCTION
Everyday we hear about a new medical craze – that wonder drug, the little pill that makes all your medical problems disappear – but many never question the potential our bodies have in fighting death and disease from within. What if we were told that there is a ready arsenal inside our bodies due to cells that are so precise and specialized that they sometimes can act as a double edge sword being both immuno-enhancing and immuno-suppressive?

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells that normally differentiate along a mesodermal lineage forming osteoblasts (bone), chondrocytes (cartilage), myocytes (muscle) and adipocytes (fat) (Figure 1). Recent scientific evidence has shown that MSCs are not limited to the genesis of mesodermal tissue, but that they can form cells of endo- and ectodermal origin (Bianco, 2001). MSCs can be found in both post natal and adult bone marrow as well as fetal organs, including, but not limited to the fetal lung and liver.

Figure 1: Mesenchymal Stem Cell Lineages

Figure 2: Hematopoietic Stem Cell Lineages with Mesenchymal (stromal) Support

MSCs are found just about everywhere in the body, but are commonly obtained from the adult bone marrow where they share residence with lymphohematopoeitic stem cells (LHSCs) (Figure 3). LHSCs surround the outer periphery of the bone marrow where oxygen levels are low while MSCs surround the central sinus where the oxygen levels are higher (Figure 4). They can be identified by positive selection for CD44, CD105, CD29 and negative selection for the hematopoietic marker, CD34 and the endothelial marker, CD31 (Rameshwar, 2006)  Although MSC are primarily found in the bone marrow, their prevalence is minimal, representing approximately 0.001-0.1% of nucleated cells (Pittenger, 1999).  MSCs express both MHC Class I and Class II markers.

Figure 3: Bone Marrow Coexistence—Mesenchymal (stromal) stem cells support hematopoietic stem cells in the bone marrow.

Figure 4: Bone Marrow Niche

HISTORY
MSCs were first distinguished by German pathologist Julius Cohnheim under the tutelage of Dr. Rudolf Virchow. Virchow was a well-known doctor who is accredited as the first doctor to recognize leukemia in 1858. He also acclaimed fame by publishing his theory on cell derivation titled Omnis cellula e cellula – “every cell originates from another cell.” In 1867, Cohnheim proved that the emigration of white blood cells is the origin of pus, the body’s inflammatory response to bacterial infection. His findings were published in Virchow’s Archiv für Pathologische Anatomie und Physiologie und für Klinische Medizin – “Archives for Pathological Anatomy and Physiology and for Clinical Medicine” – within his personal essay Ueber Entzündung und Eiterung – “About Inflammation and Sepsis.” In 1970, Alexander Friedenstein acknowledged the growth of fibroblasts in cultures of guinea pig bone marrow and spleen cells and later published his findings. This supported the idea that MSCs and their committed progenitors can form CFU-F (Colony Forming Units-Fibroblast), further proving their vital role in the body’s immune system and response to disease. He and his team were also the first to utilize in vitro culture with laboratory animal transplantation(Bianco, 2001).

ISOLATION AND EXPANSION OF MSCs
Human MSCs are able to be easily expanded and isolated for experimental use.  From freshly harvested bone marrow aspirate, one can isolate MSCs by selecting cells with a short term adherence, removing macrophages (CD14+) and red blood cells (CD45+), and selecting for CD73+ or CD49a+ cells (Boiret, 2005).  Other markers include CD105+, CD166+, CD29+, ion channels, and low affinity neuron growth factor receptor (Campagnoli, 2001, Li, 2006, Quirici, 2002). These isolated cells are morphologically similar to fibroblasts, albeit symmetric instead of asymmetric.  Since MSCs exhibit symmetric division in vitro, the cells are easily expanded.  Many laboratories are able to easily use MSCs in reproducible experiments. 

IMMUNE PROPERTIES
MSCs are very unique in terms of their functional immune properties.  In addition to their ability to generate numerous tissues, MSCs also have the ability to provide security for the cells that enter and exit the bone marrow, labeling them as the “gatekeepers” of the bone marrow.  They have the ability to maintain bone marrow homeostasis during an immune reaction thus indirectly controlling what goes in and out of the bone marrow.  Furthermore, their anatomical location surrounding the central sinus of the bone marrow allows for their “policing” of the bone marrow.  In addition, allogeneic MSCs can be transplanted due to their immunosuppressive property, making them desirable in the field of regenerative medicine (Castillo, 2006).  

As stated earlier, Cohnheim proved that the emigration of white blood cells is the origin of pus, an immunogenic response the body has to a bacterial infection. MSCs play an active role in the body’s response to foreign lymphocytes during transplantation, a response commonly called a mixed lymphocyte reaction (MLR). During MLRs the MSCs are thought to suppress alloantigens at all stages. Normal cytotoxic response involves both natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs). CTLs target foreign MHC-I cells during an immunological response. In the presence of MSCs, NK cells are suppressed and the frequency of new active CTLs in peripheral blood is lowered. These properties are important in that MSCs have demonstrated the potential to suppress Graft Versus Host Disease (GVHD), a common problem with organ transplants, especially bone marrow (Castillo, 2006).

MSC also express behavior that has been characterized as “veto-like.” This is due to the fact that they can control and override an immune response via bypassing allogenic barriers that commonly cause transplant problems (Potian, 2003). Unlike governmental voting where the majority rules, we can assume that the MSC act as the supreme-being where their indiction to the body’s natural immune response takes priority.

PLASTICITY
The plasticity of MSCs remains controversial. The definition of plasticity, where cells cross lineage barriers after being placed in a different microenvironment to form tissues other than the original intent, has two subsets. The subsets are different in that the new type of tissue formed is of a different germ layer than the original intent. New findings in this field are controversial because of the difficulty and variation in experimental methods, such as isolation techniques and the fact that currently accepted stem cell lineage theories do not allow for plasticity.

There is a good amount of evidence showing that the first subset, where mesenchymal stem cells are able to transdifferentiate into progenitors within the mesoderm germ layer, can be controlled by manipulating the microenvironment.  For example, it has been shown that rat mesenchymal stem cells genetically engineered to overexpress Akt1, a prosurvival gene, and transplanted into an ischemic rat myocardium, inhibited the process of cardiac remodeling.  The following properties were observed in the rat myocardium:  the reduction of intramyocardial inflammation, collagen deposition and cardiac myocte hypertrophy, regeneration of 80-90% of lost myocardial volume, and completely normalizing systolic and diastolic cardiac function (Mangi, 2003). In another experiment, it was shown that the injection of mesenchymal stem cells into damaged muscles leads to cells with myocyte gene expression (Ferrari, 1998). Although several experiments have shown that mesenchymal stem cells, when cultured properly and transplanted in the specific tissue in vivo, have the potential to be plastic and used for therapeutic benefit, there is the possibility that these functional properties develop due to cell fusion, macrophages which have ingested foreign proteins, or visitor cells (Holden, 2002). Therefore, these results must be interpreted with caution.

Regarding the second subset of plastic properties, where cells transdifferentiate into cells of a different germ layer, there are some reports that mesenchymal stem cells can differentiate in vitro into neuronal-type cells when transplanted into the brains of rats, whereas other cells cannot obtain this phenotype (Azizi, 1998). It is thought that if these properties do exist, they are rare.

There are many questions regarding plasticity. First of all, what is the nature of plastic properties? Does the bone marrow have primordial cells equivalent to embryonic stem cells? Also, does the bone marrow niche include uni-potential or bi-potential stem cells that give rise to particular tissues?  A recently published paper provided data that CD34- stem cells in the bone marrow have the ability to differentiate into hematopoietic and mesenchymal progenitors (Huss, 2000); therefore, do hematopoietic and mesenchymal stem cells originate from the same source? These reports indicate that the stem cell lineages might not be fully mapped and therefore provide future areas of research.

APPLICATIONS - CURRENT AND FUTURE
There are myriad therapeutic possibilities for mesenchymal stem cells, due to multi-potential properties, the ease of expansion in vitro, the allogeneic and veto properties as referenced in the Immune Properties section, and the increased potential usage in gene therapy (Minguell, 2001).

Organ Generation / Transplants
MSCs have been shown to produce cells from its own lineages, such as bone, cartilage, muscle, ligament, tendon, adipose, stroma, and from other germ layers under specific laboratory conditions, such as pancreatic, kidney, heart and neural cells. If MSCs can produce cells for a specific organ in vitro, could they be engineered to generate an actual organ in vitro? Scientists right now are trying to answer that question by learning how to manipulate the lineages MSCs express. The ability to grow organs, or even large amounts of cultured tissue, would prove extremely beneficial in many different medical and scientific fields.  For instance, in the pharmaceutical industry, new drugs and treatments could be tested on actual human cells. This could eliminate the use of animal models, cutting costs and saving the lives of numerous laboratory animals. Furthermore, if these organs could be produced by the recipient or as universally recognized tissue, rejection and Graft Versus Host Disease could be avoided. This would eliminate the need for transplantation matching, thereby rendering expensive and time-consuming transplantation lists unnecessary. 

Regarding the immune properties, there are several studies which show that a small dosage of MSCs in the area of transplantation will decrease the probability and/or severity of Graft Versus Host Disease (Rameshwar, 2006). There have been positive results from completed clinical trials studying the affect of MSCs on alleviating different types of Graft Versus Host Disease. 

It has also been shown that MSCs can home and engraft in the bone marrow.  This presents the potential therapeutic use of MSCs for autoimmune diseases (El-Badri, 2004). A murine model study has shown that MSCs can effectively interfere during an autoimmune attack in the course of experimental autoimmune encephalomyelitis, inducting T-cell anergy in the secondary lymphoid organs (Zappia, 2005). 

Cancer
Regarding cancer treatments, recent studies indicate that native MSCs prevent metastasis with neural cell adhesion molecules by stabilizing the tumor’s vessel walls. Consequently, perturbations of the MSCs would facilitate metastasis (Xian, 2006). It has also been shown that the infusion of MSCs into melanoma cells worsened the existing cancer (Djouad, 2003), probably due to angiogenic properties of the MSCs. Further, MSCs have been observed to set up pre-metastatic niches in rats when injected four weeks prior to injection of cancer cells (Steeg, 2005). 

Gene Delivery System
Mesenchymal stem cells also have the possibility to be used as a gene delivery system. For example, mesenchymal stem cells have homing systems to allow expanded stem cell populations to migrate towards sites of injury, such as CXCR4/SDF-1alpha, HGF/c-met, and MMPs (Son, 2006).

Currently, MSCs are being studied in local implantation for diseases, transplantations, use in gene therapy, and the use in tissue engineering regeneration.  Many studies indicate positive uses for MSCs, such the transplantation of mesenchymal stem cells enhancing hematopoiesis (Angelopoulou, 2003), such as enhanced myelopoiesis and karyocytopoeisis, which are thought to be derived from hematopoietic lineages only (Muguruma, 2006).  Conversely, because MSCs are involved in hematopoiesis, there is also a serious possibility of adversely affecting hematopoietic regulation and bone marrow support, which could result in multiple organ failure.

There are several ongoing trials involving the use of MSCs as therapies. A study was published in 2002 regarding six pediatric patients with osteogenesis imperfecta, a type 1 collagen defect, who received MSCs post-transplant. The patients showed improved engraftment (Horwitz, 2002). As of November 2006, recent human clinical trials closed to additional enrollement for the use of MSCs in heart failure, Crohn’s Disease, post-menisectomy treatment, regeneration of periodontal tissue, Graft Versus Host Disease, and cancer.  There is also a trial geared towards the treatment of multiple sclerosis which is not yet recruiting patients.

Many scientists believe in the future use of MSCs in the clinic.   However, until we can study and control the affect of MSCs on the hematopoietic system and the controlled differentiation into desired lineages, the failure of MSC treatments might always be a possibility.

References

Angelopoulou M, Novelli E, Grove JE, Rinder HM, Civin C, Cheng L, Krause DS (2003) Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice. Exp Hematol 31:413-20.

Azizi SA, Stokes D, Augelli BJ, DiGirolamo C, Prockop DJ (1998) Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats-similarities to astrocyte grafts.  Proc Natl Acad Sci U S A 95:3908-13.

Bianco P, Riminucci M, Gronthos S, Robey PG (2001) Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19:180-92.

Boiret N, Rapatel C, Veyrat-Masson R, Guillouard L, Guerin JJ, Pigeon P, Descamps S, Boisgard S, Berger MG (2005) Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow.  Exp Hematol 33:219-25.

Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM (2001) Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 98:2396-402.

Castillo M, Liu K, Bonilla L, Rameshwar P (2006) The immune properties of mesenchymal stem cells. In Press

Djouad F, Plence P, Bony C, Tropel P, Apparailly F, Sany J, Noel D, Jorgensen C (2003) Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood 102:3837-44.

El-Badri NS, Maheshwari A, Sanberg PR (2004) Mesenchymal stem cells in autoimmune disease. Stem Cells Dev 13:463-72.

Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F (1998) Muscle regeneration by bone marrow-derived myogenic progenitors.  Science 279: 1528-30.

Holden C, Vogel G (2002) Plasticity: Time for a Reappraisal?.  Science 296:2126.

Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, Muul L, Hofmann T (2002) Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A 99:8932-7.

Huss R (2000) Perspectives on the Morphology and Biology of CD34-Negative Stem Cells. J Hematotherapy & Stem Cell Res 9:783-93.

Minguell J, Erices A, Conget P (2001) Mesenchymal Stem Cells. Expt Biol and Med 226:507-20.

Li GR, Deng XL, Sun H, Chung SS, Tse HF, Lau CP (2006) Ion channels in mesenchymal stem cells from rat bone marrow. Stem Cells 24:1519-28.

Mangi AA, Noiseux N, Kong D, He H, Rezvani M, Ingwall JS, Dzau VJ (2003) Mesenchymal Stem Cells Modified with Akt prevent Remodeling and Restore Performance of Infarcted Hearts. Nature Med 9:1195-201.

Muguruma Y, Yahata T, Miyatake H, Sato T, Uno T, Itoh J, Kato S, Ito M, Hotta T, Ando K (2006) Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment. Blood 107: 1878-87.

Pittenger MF, Mackay Am, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143-7.

Potian J, Aviv H, Ponzio N, Harrison J, Rameshwar P (2003) Veto-like activity of mesenchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens. J Immunol 171:3426-34.

Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C, Deliliers GL (2002) Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies.  Exp Hematol 30:783-91.

Son BR, Marquez-Curtis LA, Kucia M, Wysoczynski M, Turner AR, Ratajczak J, Ratajczak MZ, Janowska-Wieczorek A (2006) Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells 24:1254-64.

Steeg PS (2005) Cancer biology: emissaries set up new sites.  Nature 438:750-1.

Zappia E, Casazza S, Pedemonte E, Benvenuto F, Bonanni I, Gerdoni E, Giunti D, Ceravolo A, Cazzanti F, Frassoni F, Mancardi G, Uccelli A (2005) Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 106:1755-61.

Online Encyclopedia Entry for Julius Cohnheim: http://www.jewishencyclopedia.com/view_friendly.jsp?artid=660&letter=C

Acknowledgements
This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey:

Tara Gooen, Joel Schneider, and James Sturzione (in alphabetical order) Fall 2006
Teaching Assistant: Kathy Trzaska